A saussurea cell culture and its use in the preparation of a medicament for the treatment of ovarian dysfunction

Abstract

The embodiment of the specification provides a saussurea cell culture and a use thereof in preparation of a medicine for treating ovarian function impairment, wherein the saussurea cell culture is a cell culture dry product obtained by amplification culture, collection and drying of in-vitro cultured cells of a saussurea plant; and the cell culture dry product contains at least two of chlorogenic acid, syringa, 1,5-O-dicaffeoylquinic acid and rutin.

Description

Technical Field

[0001] This specification relates to the fields of medicine and biotechnology, and in particular to a snow lotus cell culture and its use in the preparation of a medicament for treating ovarian dysfunction. Background Technology

[0002] Premature ovarian decline in women before the age of 40 often manifests as a decrease in the number of follicles, hormonal imbalances, and reduced fertility. Chemotherapy drugs (such as alkylating agents like cyclophosphamide) are a common cause of ovarian damage, damaging oocytes and ovarian granulosa cells, promoting follicular atresia, and disrupting steroid hormone synthesis. This type of chemotherapy-related ovarian dysfunction has become a significant clinical problem affecting female patients' fertility and endocrine health. Currently, clinical intervention mainly involves hormone replacement therapy or assisted reproductive technologies, but the efficacy is limited and it is difficult to fundamentally restore ovarian reserve and function. Therefore, there is an urgent need to develop therapeutic drugs that target the pathological aspects of ovarian damage and improve follicular reserve and endocrine function.

[0003] Snow lotus contains various active ingredients such as caffeoylquinic acid, phenylpropanoid glycosides, and flavonoids, which have certain protective potential for ovarian function. However, wild snow lotus is greatly affected by factors such as its place of origin, harvesting season, and ecological environment, resulting in unstable components and significant fluctuations in efficacy, making it difficult to produce standardized drugs with controllable quality.

[0004] Therefore, it is necessary to provide a snow lotus cell culture and its use in the preparation of drugs for treating ovarian dysfunction, in order to improve chemotherapy-related ovarian dysfunction, enhance ovarian reserve and endocrine function recovery, reduce the proportion of follicular atresia, and reduce dependence on wild snow lotus resources, thereby achieving quality control and batch consistency. Summary of the Invention

[0005] This specification provides one or more embodiments for the use of a standardized snow lotus cell culture in the preparation of a drug for treating chemotherapy-related ovarian dysfunction. The standardized snow lotus cell culture is a dried cell culture obtained by expanding, harvesting, and drying in vitro cultured cells of the genus Saussurea. The dried cell culture contains at least two of chlorogenic acid, syringin, 1,5-O-dicaffeoylquinic acid, and rutin.

[0006] In some embodiments, the snow lotus plant is Saussurea involucrata (Kar. etKir.) Sch.-Bip.; the dried cell culture contains chlorogenic acid, syringin, 1,5-O-dicaffeoylquinic acid and rutin.

[0007] In some embodiments, the chemotherapy-related ovarian dysfunction is induced by cyclophosphamide or its active metabolites; the drug is administered after cyclophosphamide administration has ended and ovarian damage has already occurred.

[0008] In some embodiments, the therapeutic effects of the drug include: compared with an untreated chemotherapy-related ovarian dysfunction model, one or more of the following in the serum of the subjects after administration: increased levels of anti-Müllerian hormone (AMH), increased levels of estradiol (E2), and decreased levels of follicle-stimulating hormone (FSH).

[0009] In some embodiments, the therapeutic effects of the drug further include: an increase in the absolute number of primordial follicles, primary follicles, or secondary follicles in ovarian tissue sections of subjects after administration, and / or a decrease in the proportion of atretic follicles to the total number of follicles, compared to an untreated chemotherapy-related ovarian dysfunction model.

[0010] In some embodiments, the therapeutic effects of the drug further include changes in the ovarian tissue of the subjects after administration, compared to an untreated chemotherapy-related ovarian dysfunction model, including at least one of the following: upregulation of mRNA expression levels of Cyp17a1, Cyp19a1, Hsd17b1, or anti-Müllerian hormone; decreased mRNA expression levels of acute regulatory steroid protein (Star) or scavenger receptor class B member 1 (Scarb1); or, reversion of pregnenolone, desmosterol, 7-ketocholesterol, 7α,25-dihydroxycholesterol, 25-hydroxycholesterol, corticosterone, testosterone, progesterone, androstenedione, 17α-hydroxyprogesterone, or 17β-estradiol to normal physiological levels.

[0011] In some embodiments, the drug is an oral preparation selected from tablets, capsules, granules, pills, powders, oral liquids, suspensions, and lyophilized powders reconstituted before oral administration; the unit dose of the preparation contains 50 to 500 mg of the dried cell culture.

[0012] This specification provides one or more embodiments of a pharmaceutical composition comprising a standardized Saussurea involucrata cell culture and pharmaceutically acceptable excipients; the standardized Saussurea involucrata cell culture is a dried cell culture obtained by expanding, harvesting, and drying in vitro cultured cells of Saussurea involucrata (Kar. et Kir.) Sch.-Bip.; the dried cell culture contains at least two of chlorogenic acid, syringin, 1,5-O-dicaffeoylquinic acid, and rutin.

[0013] In some embodiments, the pharmaceutical composition is used to treat chemotherapy-related ovarian dysfunction induced by cyclophosphamide or its active metabolites; the pharmaceutical composition is a tablet, capsule, granule, pill, powder, oral liquid, suspension, or lyophilized powder reconstituted before oral administration.

[0014] This specification provides one or more embodiments of a standardized method for preparing a Saussurea cell culture, comprising: obtaining sterile explants or callus tissue of Saussurea plants; establishing and expanding in vitro cultured cells; harvesting cells and removing residual culture medium; drying and pulverizing to obtain a dried cell culture product; wherein the dried cell culture product contains at least two of chlorogenic acid, syringin, 1,5-O-dicaffeoylquinic acid, and rutin.

[0015] In some embodiments, the establishment and expansion of in vitro cultured cells includes: seeding 20-30 g of fresh cell weight per liter of culture medium, culturing at 23-27 °C and 100-120 r / min for 12-16 days, and scaling up through at least two stages of culture in shake flasks, seed tanks, and production tanks.

[0016] This specification provides one or more embodiments of a standardized method for quality control of snow lotus cell cultures, comprising: using high performance liquid chromatography (HPLC) at a detection wavelength of 265 nm to compare the retention times of each characteristic peak in the chromatogram of the test sample with the retention times of reference peaks of chlorogenic acid, syringin, 1,5-O-dicaffeoylquinic acid, and rutin; using LC-HRMS / MS to confirm the structure of snow lotus lactone; and determining that batches containing at least two of chlorogenic acid, syringin, 1,5-O-dicaffeoylquinic acid, and rutin, and whose relative deviations of the retention times of each characteristic peak from the corresponding reference peaks do not exceed 2.0%, are qualified as meeting the requirements for characteristic component identification.

[0017] In some embodiments, the method further includes process control of live cell samples before harvesting, the process control including: using GAPDH2 as an internal reference gene, qRT-PCR is used to detect the relative expression levels of at least one of the following genes: hydroxycinnamoyl-CoA quinoline hydroxycinnamoyltransferase (HQT), shikimate O-hydroxycinnamoyltransferase (HCT), flavonoid synthase (FNS), UDP-glycosyltransferase (UGT), O-methyltransferase (OMT), chalcone synthase (CHS), flavanone 3-hydroxylase (F3H), and flavonoid 3'-hydroxylase (F3'H), as well as at least two of the sesquiterpene lactone candidate genes with sequences as shown in SEQ ID NO: 1-24, to assist in characterizing the culture stage and batch consistency.

[0018] This specification provides one or more embodiments of a method for preparing enriched fractions of snow lotus cell culture, comprising the following steps: preparing snow lotus (Saussurea involucrata (Kar. et Kir.) Sch.-Bip.) The dried product of in vitro cultured cells was extracted twice by reflux at 80±2 °C for 1 h each time, with 10 mL of 50% ethanol aqueous solution per gram of dried product. The filtrates were combined and filtered while hot. The combined filtrate was concentrated under reduced pressure at ≤60 °C and -0.08 to -0.06 MPa to a relative density of 1.10 to 1.20 (measured at 60 °C) to obtain a concentrate. The concentrate was loaded onto a D101 macroporous adsorption resin column at a flow rate not exceeding 1 column volume / hour. The column was first eluted with 3 column volumes of purified water to remove impurities, and then eluted with 3 column volumes of 30% ethanol aqueous solution. The 30% ethanol eluent was collected and concentrated under reduced pressure at ≤60 °C and then freeze-dried to obtain an enriched fraction containing 1,5-O-dicaffeoylquinic acid and rutin. Attached Figure Description

[0019] This specification will be further described by way of exemplary embodiments, which will be described in detail with reference to the accompanying drawings. These embodiments are not limiting; in these embodiments, the same reference numerals denote the same structures, wherein: Figure 1 This is an exemplary flowchart of a standardized method for preparing snow lotus cell cultures according to some embodiments of this specification; Figure 2 HPLC characteristic component identification chromatograms of standardized snow lotus cell cultures; Figure 3 A schematic diagram showing the relative expression levels of HQT, HCT, FNS, UGT, OMT, CHS, F3H, and F3'H in callus tissue, suspension culture, scale-up culture, and harvest period; Figure 4 A schematic diagram of the biosynthetic pathway and candidate genes of sesquiterpene lactones in Saussurea involucrata. Figure 5 A schematic diagram showing the relative expression patterns of six candidate genes for sesquiterpene lactone biosynthesis in callus tissue, early suspension culture, logarithmic growth phase of scale-up culture, and live cells at harvest time; Figure 6 Flowchart for cyclophosphamide modeling, SIC treatment, fertility monitoring, and sampling; Figure 7 This is a schematic diagram illustrating the effects of SIC on rat serum E2, AMH, and FSH, as shown in some examples of this specification. Figure 8A HE staining images of the normal control group, model group, E2 group, SIC-L group, SIC-M group and SIC-H group; Figure 8BA schematic diagram showing the counting of atretic follicles, secondary follicles, primordial follicles, and primary follicles; Figure 9 This is a schematic diagram showing the effect of SIC on the relative expression levels of Cyp17a1, Cyp19a1, Hsd17b1, Star, Scarb1 and Amh in ovarian tissue. Figures 10A-10B These are schematic diagrams illustrating the effects of SIC on pregnenolone, desmosterol, 7-ketocholesterol, 7α,25-dihydroxycholesterol, 25-hydroxycholesterol, corticosterone, testosterone, progesterone, androstenedione, 17α-hydroxyprogesterone, and 17β-estradiol. Detailed Implementation

[0020] To more clearly illustrate the technical solutions of the embodiments in this specification, the accompanying drawings used in the description of the embodiments will be briefly introduced below. Obviously, the drawings described below are merely some examples or embodiments of this specification. For those skilled in the art, these drawings can be applied to other similar scenarios without creative effort. Unless obvious from the context or otherwise specified, the same reference numerals in the drawings represent the same structures or operations.

[0021] As indicated in this specification and claims, unless the context clearly indicates otherwise, the words "a," "an," "an," and / or "the" do not specifically refer to the singular and may also include the plural. Generally speaking, the terms "comprising" and "including" only indicate the inclusion of expressly identified steps and elements, which do not constitute an exclusive list, and the method or apparatus may also include other steps or elements.

[0022] This specification provides an example of the use of a standardized snow lotus cell culture in the preparation of a medicament for treating chemotherapy-related ovarian dysfunction. In some embodiments, the standardized snow lotus cell culture is a dried cell culture obtained by expanding, harvesting, and drying in vitro cultured cells of the genus *Saussurea*. In some embodiments, the dried cell culture contains at least two of chlorogenic acid, syringin, 1,5-O-dicaffeoylquinic acid, and rutin.

[0023] Standardized cell culture of Saussurea involucrata (SIC) refers to dried Saussurea cell culture obtained from plants of the Saussurea genus through in vitro cell culture technology, possessing clearly defined chemical quality markers and batch-to-batch consistency. In some embodiments, standardized Saussurea cell culture is obtained from sterile explants, callus tissue, suspension culture cells, or cell clusters of Saussurea genus plants through in vitro culture. Further instructions on obtaining standardized Saussurea cell culture can be found in [link to relevant documentation]. Figure 1 .

[0024] In some embodiments, the chemical characteristics of SIC can be characterized by high-performance liquid chromatography (HPLC). In some embodiments, HPLC is used at a detection wavelength of 265 nm to compare the chromatogram of the SIC test sample with that of the reference sample. At least two characteristic components from chlorogenic acid, syringin, 1,5-O-dicaffeoylquinic acid, and rutin are detected in the SIC test sample. That is, the dried SIC contains at least two of chlorogenic acid, syringin, 1,5-O-dicaffeoylquinic acid, and rutin.

[0025] Reference standards are known chemical standard substances used in analytical methods such as high-performance liquid chromatography (HPLC). Reference standards have a defined chemical structure, source, and purity, and are used for the qualitative identification and / or quantitative analysis of corresponding characteristic components in test samples. Reference standards include standards for characteristic components such as chlorogenic acid, syringin, 1,5-O-dicaffeoylquinic acid, and rutin. Reference standards can be obtained commercially or prepared and calibrated in-house.

[0026] Reference standards from the same batch refer to reference solutions prepared under the same conditions as the test solution and injected into the same analytical sequence in the same analytical experiment. Using reference standards from the same batch for retention time comparison can reduce the impact of minor fluctuations in retention time between different analytical batches caused by systemic factors such as chromatographic column, mobile phase, instrument status, and environmental conditions, thereby ensuring the accuracy and repeatability of characteristic peak attribution.

[0027] In some embodiments, the retention times of each characteristic peak in the chromatogram of the SIC test sample are compared with the retention times of the corresponding component reference peak prepared in the same batch. If the relative deviation between the two does not exceed 2.0%, the component is considered to be detected in the test sample.

[0028] In some embodiments, SIC also has a characteristic spectrum centered on lactone chemical markers. Lactone chemical markers are represented by yalian lactone, and may further include at least one of dehydroaurolide lactone, zearalenone, ergosterol, and atractylodes lactone I. To avoid confusion with compounds of the same name, "yalian lactone" in this specification refers to Xuelianlactone. When establishing specific quality standards, unique identification should be performed using the name, structural formula, CAS number, retention time, and high-resolution mass spectrometry fragment information of certified reference standards. Certified reference standards refer to those provided by authoritative institutions such as the China National Institutes for Food and Drug Control, the United States Pharmacopeia (USP), and the European Pharmacopoeia (EP), or those prepared by standard substance manufacturers that meet the requirements of ISO 17034 and are accompanied by a certificate. When certified reference standards are unavailable, high-purity reference standards with confirmed structure and a purity of not less than 98% can be used, combined with high-resolution mass spectrometry, nuclear magnetic resonance, and other methods for identification.

[0029] Ovarian dysfunction refers to an abnormality or decline in at least one of the following: ovarian reserve, follicular development, ovarian endocrine function, or ovarian tissue structure. Ovarian dysfunction can manifest as one or more of the following: decreased ovarian reserve, reduced number or quality of follicles, increased follicular atresia, decreased estrogen levels, abnormally elevated gonadotropin levels, or impaired fertility. In some embodiments, ovarian dysfunction is drug-induced ovarian dysfunction. In some embodiments, drug-induced ovarian dysfunction can be antitumor drug-induced ovarian dysfunction. In some embodiments, the antitumor drug is cyclophosphamide or its active metabolite.

[0030] In some preferred embodiments, the plant in the genus Saussurea is the snow lotus (Saussurea involucrata (Kar. etKir.) Sch.-Bip.).

[0031] In some embodiments, HPLC is used at a detection wavelength of 265 nm to compare the chromatogram of the SIC test sample with that of the reference sample. The SIC test sample simultaneously detects four components: chlorogenic acid, syringin, 1,5-O-dicaffeoylquinic acid, and rutin, and the relative deviation of the retention time of the characteristic peak of each component from the corresponding reference peak does not exceed 2.0%. That is, the dried SIC contains chlorogenic acid, syringin, 1,5-O-dicaffeoylquinic acid, and rutin.

[0032] In some embodiments, chemotherapy-related ovarian dysfunction is induced by cyclophosphamide or its active metabolites. The drug is administered after cyclophosphamide administration has ceased and ovarian damage has already occurred.

[0033] Cyclophosphamide is an alkylating agent antitumor drug that can be metabolized in vivo into active metabolites that damage oocytes and ovarian granulosa cells, promote follicular atresia, and disrupt steroid hormone synthesis. Therefore, it is often used to establish animal models of chemotherapy-related ovarian dysfunction.

[0034] A drug is a substance or preparation used to improve or treat ovarian insufficiency, particularly chemotherapy-related ovarian dysfunction, which contains a therapeutically effective amount of standardized snow lotus cell culture (SIC) as the active ingredient and may optionally contain one or more pharmaceutically acceptable carriers, excipients or excipients.

[0035] Ovarian damage has already occurred when, after the cyclophosphamide modeling treatment, the animal exhibits ovarian structural and / or functional abnormalities, such as a decrease in the number of follicles, an increase in atretic follicles, a decrease in serum AMH or E2, or an increase in FSH, at which point SIC administration is considered therapeutic rather than prophylactic. It should be noted that the cyclophosphamide model is used to provide specific efficacy support and validation of the uses described in the examples of this specification, and should not be construed as a general limitation on the use of the ovarian dysfunction described in the examples of this specification. For ovarian dysfunction caused by different etiologies or different pathological mechanisms, the specific therapeutic effects can still be further validated based on the corresponding models or clinical studies.

[0036] In some embodiments, the therapeutic effects of the drug include: one or more of the following in the serum of subjects after administration: elevated levels of anti-Müllerian hormone (AMH), elevated levels of estradiol (E2), and decreased levels of follicle-stimulating hormone (FSH) compared to an untreated chemotherapy-associated ovarian dysfunction model.

[0037] Test subjects refer to individuals treated with standardized snow lotus cell culture (SIC) or its pharmaceutical composition, including mammals. Mammals include, but are not limited to, laboratory animals such as rats, mice, rabbits, dogs, and monkeys.

[0038] In the embodiments of this specification, the test subjects were female SD rats. A chemotherapy-related ovarian dysfunction model was established by administering cyclophosphamide, and SIC was administered after the damage occurred to evaluate its efficacy. Therefore, the test subjects in the embodiments specifically refer to individuals within this rat model.

[0039] The untreated chemotherapy-related ovarian dysfunction model refers to control animals that received the same modeling treatment but were not given SIC, or were only given solvent or blank treatment.

[0040] It should be understood that the uses described in the embodiments of this specification are not limited to the animal species used in the embodiments. Provided that relevant preclinical or clinical data is available to support this, the subjects may be expanded to other mammals or human patients, especially female patients requiring treatment for chemotherapy-related ovarian dysfunction.

[0041] Furthermore, in the embodiments of this specification, when biological samples (such as serum and ovarian tissue) of the test subjects are involved, the detection indicators are all based on the samples of the test subjects themselves, and are compared with the corresponding samples of the untreated chemotherapy-related ovarian function damage model.

[0042] Anti-Müllerian hormone (AMH) is a glycoprotein hormone secreted by ovarian granulosa cells. Its serum level is closely related to ovarian reserve and is an important indicator for evaluating ovarian function. Estradiol (E2) is the main estrogen secreted by the ovaries and reflects ovarian endocrine function. Follicle-stimulating hormone (FSH) is a gonadotropin secreted by the pituitary gland. When ovarian function declines, FSH usually rises abnormally due to weakened negative feedback. Elevated AMH, elevated E2, and decreased FSH collectively indicate improved ovarian reserve and endocrine function.

[0043] In some embodiments, the therapeutic effects of the drug also include: an increase in the absolute number of primordial follicles, primary follicles, or secondary follicles in ovarian tissue sections of subjects after administration, and / or a decrease in the proportion of atretic follicles to the total number of follicles, compared to an untreated chemotherapy-related ovarian dysfunction model.

[0044] In some embodiments, the therapeutic effects of the drug further include changes in the ovarian tissue of the subjects after administration, compared to an untreated chemotherapy-related ovarian dysfunction model, including at least one of the following: upregulation of mRNA expression levels of Cyp17a1, Cyp19a1, Hsd17b1, or anti-Müllerian hormone; decreased mRNA expression levels of acute regulatory steroid protein (Star) or scavenger receptor class B member 1 (Scarb1); or, reversion of pregnenolone, desmosterol, 7-ketocholesterol, 7α,25-dihydroxycholesterol, 25-hydroxycholesterol, corticosterone, testosterone, progesterone, androstenedione, 17α-hydroxyprogesterone, or 17β-estradiol to normal physiological levels.

[0045] The term "reverting to normal physiological levels" refers to the level of metabolites tending to be similar to that of undamaged animals (normal control group), without requiring the metabolite levels to fully recover to normal values.

[0046] The aforementioned changes in genes and metabolites are used to illustrate the association between SIC and the restoration of the ovarian steroid production network, and do not indicate that SIC directly binds to or directly regulates a single target, nor do they limit the mechanism of action to a single mechanism.

[0047] In some embodiments, the drug is an oral preparation selected from tablets, capsules, granules, pills, powders, oral liquids, suspensions, and lyophilized powders reconstituted before oral administration. In some embodiments, a unit dose of the preparation contains 50–500 mg of dried cell culture material.

[0048] Unit dose refers to the labeled amount of active ingredient contained in a single smallest unit of administration (such as a tablet, a pill, a sachet, a bottle, a pellet, etc.).

[0049] This specification also provides a pharmaceutical composition comprising a standardized Saussurea involucrata cell culture and pharmaceutically acceptable excipients. In some embodiments, the standardized Saussurea involucrata cell culture is a dried cell culture obtained by expanding, harvesting, and drying in vitro cultured Saussurea involucrata (Kar. et Kir.) Sch.-Bip. The standardized Saussurea involucrata cell culture contains at least two of chlorogenic acid, syringin, 1,5-O-dicaffeoylquinic acid, and rutin.

[0050] In some embodiments, the pharmaceutical composition is used to treat chemotherapy-related ovarian dysfunction induced by cyclophosphamide or its active metabolites. In some embodiments, the pharmaceutical composition is in the form of tablets, capsules, granules, pills, powders, oral liquids, suspensions, or lyophilized powder reconstituted before oral administration. Further details regarding the pharmaceutical composition can be found in Example 9.

[0051] In some embodiments, the standardized Saussurea involucrata cell culture in the pharmaceutical composition is present in a concentration of 100 mg per unit dose, calculated as SIC (saturated calcium carbonate) dried matter. The unit dose may be adjusted for clinical use based on dosage conversion and formulation validation. When the pharmaceutical composition is administered orally to subjects, the daily dose, calculated as SIC dried matter, is 100–420 mg / kg body weight, preferably 210–420 mg / kg body weight. The above daily dose range is determined based on pharmacodynamic data obtained from a cyclophosphamide-induced rat ovarian dysfunction model and is used to support the pharmaceutical use of the pharmaceutical composition. It is not directly equivalent to the human clinical dose, but it can provide a basis for dosage conversion.

[0052] This specification provides a standardized method for preparing a Saussurea involucrata cell culture, comprising the following steps: obtaining sterile explants or callus tissue from Saussurea plants; establishing and expanding in vitro cultured cells; harvesting cells and removing residual culture medium; and drying and pulverizing to obtain a dried cell culture product. In some embodiments, the dried cell culture product contains at least two of chlorogenic acid, syringin, 1,5-O-dicaffeoylquinic acid, and rutin.

[0053] In some embodiments, establishing and expanding in vitro cultured cells includes: seeding 20–30 g of fresh cell weight per liter of culture medium, culturing at 23–27 °C and 100–120 r / min for 12–16 days, and scaling up through at least two stages of culture in shake flasks, seed tanks, and production tanks. Further details on the preparation method of standardized snow lotus cell cultures can be found at [link to relevant documentation]. Figure 1 and Figure 2 .

[0054] This specification also provides a standardized quality control method for snow lotus cell cultures, comprising: using high-performance liquid chromatography (HPLC) at a detection wavelength of 265 nm to compare the retention times of each characteristic peak in the chromatogram of the test sample with the retention times of reference peaks of chlorogenic acid, syringin, 1,5-O-dicaffeoylquinic acid, and rutin; using LC-HRMS / MS to confirm the structure of snow lotus lactone; and determining that batches containing at least two of chlorogenic acid, syringin, 1,5-O-dicaffeoylquinic acid, and rutin, and whose relative deviations of the retention times of each characteristic peak from the corresponding reference peaks do not exceed 2.0%, are qualified as meeting the requirements for characteristic component identification.

[0055] In some embodiments, the quality control method for standardized snow lotus cell cultures further includes: using GAPDH2 as an internal reference gene, and employing qRT-PCR to detect the relative expression levels of at least one of the following genes: hydroxycinnamoyl-CoA quinoline hydroxycinnamoyltransferase (HQT), shikimate O-hydroxycinnamoyltransferase (HCT), flavonoid synthase (FNS), UDP-glycosyltransferase (UGT), O-methyltransferase (OMT), chalcone synthase (CHS), flavanone 3-hydroxylase (F3H), and flavonoid 3'-hydroxylase (F3'H), as well as at least two of the sesquiterpene lactone candidate genes with sequences as shown in SEQ ID NO: 1–24, to assist in characterizing the culture stage and batch consistency.

[0056] This specification also provides a method for preparing the enriched fraction of Saussurea involucrata cell culture, comprising the following steps: The dried in vitro cultured cells of Saussurea involucrata (Kar. et Kir.) Sch.-Bip. are extracted twice by reflux at 80±2 °C for 1 h each time, with 10 mL of 50% ethanol aqueous solution per gram of dried product. The filtrates are then combined. The combined filtrate is concentrated under reduced pressure at ≤60 °C and -0.08 to -0.06 MPa to a relative density of 1.10 to 1.20 to obtain a concentrated solution. The concentrated solution is loaded onto a D101 macroporous adsorption resin column at a flow rate not exceeding 1 column volume / hour. The column is first eluted with 3 column volumes of purified water to remove impurities, then eluted with 3 column volumes of 30% ethanol aqueous solution, and the 30% ethanol eluent is collected. The 30% ethanol eluent is then concentrated at ≤60 °C. After concentration under reduced pressure at ℃, the fraction was freeze-dried to obtain an enriched fraction containing 1,5-O-dicaffeoylquinic acid and rutin.

[0057] Figure 1 This is an exemplary flowchart illustrating a standardized method for preparing snow lotus cell cultures according to some embodiments of this specification. Figure 1 As shown, process 100 includes the following steps.

[0058] Step 110: Obtain sterile explants or callus tissue from the Saussurea genus.

[0059] In some embodiments, the preferred species of the genus Saussurea is Saussurea involucrata (Kar. etKir.) Sch.-Bip.

[0060] Plants of the genus *Saussurea* should be botanically identified by qualified institutions (such as the herbarium of the Institute of Botany, Chinese Academy of Sciences, or the National Herbarium of China). After identification, a supporting specimen should be retained, noting the person who identified the plant, the date of identification, and the specimen number to ensure traceability.

[0061] In some embodiments, the initial explants of Saussurea species are selected from botanically identified Saussurea leaves, petioles or root segments, preferably root segments, petioles or leaves of sterile seedlings obtained from surface-sterilized seeds.

[0062] Obtaining sterile explants of the genus *Saussurea* involved the following steps: before operation, the laminar flow hood was wiped with 75% ethanol and irradiated with ultraviolet light for 30 min, then the ultraviolet light was turned off and the hood was ventilated for 10 min; the directly collected tissues were first washed with running water for 20 min, then treated with 70% ethanol for 30 s in the laminar flow hood, and then treated with 2% sodium hypochlorite solution for 10 min (seeds were treated with 70% ethanol for 30 s, and then treated with 2% sodium hypochlorite solution for 15 min); after treatment, all tissues were rinsed 5 times with sterile water, blotted dry with sterile filter paper, and then cut into 2-3 mm pieces before inoculation (both cutting and inoculation were performed using sterile forceps and scalpels).

[0063] In some embodiments, obtaining callus from *Saussurea* species includes: inoculating sterile explants into an induction medium, controlling hormone, temperature, and light conditions to obtain stable callus. The medium formulation includes: callus induction on MS solid medium at pH 5.6–5.8; root or petiole explants using 0.3 mg / L 6-benzyladenine and 1.0 mg / L naphthaleneacetic acid; and leaf explants using 0.5 mg / L 6-benzyladenine and 1.5 mg / L naphthaleneacetic acid. Culture conditions include: dark culture at 25 ± 2 °C.

[0064] Step 120: Establish and expand in vitro cultured cells.

[0065] In some embodiments, cell lines may be screened before establishing and expanding in vitro cultured cells. Screening cell lines includes selecting for stable cell lines, assessing morphology, viability, and sterility, and initially screening for characteristic metabolites or target activities.

[0066] When screening cell lines, stable callus tissue obtained by induction is subcultured every 16-18 days, with subculture blocks having a diameter of 2-3 mm. During subculture, vigorous, compact, and non-water-soaked cell clusters are preferred for passage. Color is only used as an auxiliary observation indicator and is not the main basis for judging the state of callus tissue.

[0067] The proliferating cell lines were screened, including morphological observation, viability testing, and sterility checks, and preliminary screening for characteristic metabolites or target active ingredients. The screening criteria for stable cell lines were: at least 10 consecutive subcultures; using the last three generations of culture as the subjects, the fresh weight gain fold, total flavonoid content, and the HPLC peak areas of chlorogenic acid, syringin, 1,5-O-dicaffeoylquinic acid, and rutin were measured, and the relative standard deviations (RSDs) of the above indicators were calculated. When all RSDs did not exceed 15%, and the chromatographic peaks of the four characteristic components—chlorogenic acid, syringin, 1,5-O-dicaffeoylquinic acid, and rutin—were consistently detectable, the cell lines were determined to be stable. The HPLC detection methods for the four characteristic components—chlorogenic acid, syringin, 1,5-O-dicaffeoylquinic acid, and rutin—are described in step 140.

[0068] In some embodiments, establishing and expanding in vitro cultured cells includes inoculating a screened stable cell line into MS liquid medium to establish a suspension culture system. The MS liquid medium contains 0.3 mg / L 6-benzyladenine and 1.0 mg / L naphthaleneacetic acid, without added agar. The culture conditions are as follows: inoculum size 20–30 g fresh cell weight / L, liquid volume in shake flasks 25% of the container volume, culture temperature 25±2 ℃, shaker speed 110±10 r / min, illumination 12 h white light daily (1000–2000 lx), and culture period 12–16 days.

[0069] After the above-described culture, a stable amplified culture is harvested. The stable amplified culture should simultaneously meet the following conditions: the cells have regular morphology and dense cytoplasm under a microscope; the proportion of viable cells stained with trypan blue is ≥85%; the fresh weight reaches 5 to 10 times the initial inoculum; the culture medium is free of abnormal turbidity, and the smears show no microbial contamination under a microscope.

[0070] In some embodiments, establishing and expanding in vitro cell culture includes: seeding 20–30 g of fresh cell weight per liter of culture medium, culturing for 12–16 days at 23–27 °C and 100–120 r / min, and scaling up through at least two stages of culture in shake flasks, seed tanks, and production tanks. An exemplary scale-up culture includes: scaling up a stable expanded culture obtained through suspension culture in the order of shake flasks, seed tanks, intermediate tanks, and production tanks. The working volumes of each culture vessel are: 0.15 L for shake flasks, 3 L or 5 L for seed tanks, 35 L or 50 L for intermediate tanks, and 350 L or 500 L for production tanks; the inoculum size for each stage is 10% of the working volume of that stage. Temperature, pH, aeration, stirring speed, and dissolved oxygen are controlled during the culture process to ensure batch-to-batch stability and consistency. The culture temperature was maintained at 25±2 ℃, and the pH was controlled at 5.6–5.8. The stirring speeds of the seed tank, intermediate tank, and production tank were set at 80±5 r / min, 70±5 r / min, and 50–60 r / min, respectively. The aeration rate was 0.3–0.5 vvm, and dissolved oxygen was maintained at 20%–50% air saturation. Samples were taken every 2 days to monitor temperature, pH, stirring speed, aeration rate, dissolved oxygen, cell morphology, viability, fresh weight, and sterility.

[0071] Step 130: Harvest cells and remove culture medium residue.

[0072] When the culture reaches the harvest endpoint, collect the cell culture. The harvest endpoint is determined based on a combination of culture period, cell fresh weight, and viability: the culture time is 12–16 days, and two consecutive samples taken two days apart show a cell fresh weight increase of less than 10%, or cell viability decreases from an initial >90% to 85%–90%. Remove the culture medium by filtration or centrifugation, and wash the cells twice quickly with purified water. Record the harvest batch and yield.

[0073] In some embodiments, during steps 110-130, live cell samples can be collected at the callus stage, early suspension culture stage, logarithmic growth phase of scale-up culture, and harvesting stage, respectively, and gene expression can be detected using qRT-PCR. This detection is used to assist in tracking cell metabolic status and batch consistency, and is only used as an auxiliary monitoring tool during the culture process, not for final product release inspection.

[0074] In some embodiments, gene expression detection methods include detecting the expression levels of eight genes, namely HQT, HCT, FNS, UGT, OMT, CHS, F3H, and F3'H. These genes mainly target the phenylpropanoid / flavonoid metabolic pathway and are related to the biosynthesis of four characteristic components (chlorogenic acid, syringin, 1,5-O-dicaffeoylquinic acid, and rutin) by HPLC. For specific detection methods, please refer to Example 1.

[0075] In some embodiments, the gene expression detection method includes: detecting the expression levels of six candidate genes for sesquiterpene lactones, wherein the candidate genes point to the metabolic pathway of sesquiterpene lactones and are related to the biosynthesis of lactone chemical markers such as yahenolide. For specific detection methods, please refer to Example 2.

[0076] The above gene expression detection results are used for status monitoring during the culture process and to assist in judging batch-to-batch consistency, and are not used as a substitute indicator for the release inspection of dried finished products.

[0077] Step 140: Dry and pulverize to obtain dried cell culture.

[0078] The harvested and cleaned cell cultures are dried using methods such as freeze-drying, vacuum drying, or low-temperature drying at no more than 60 °C to obtain a dried product. The dried product is then pulverized so that it all passes through a 60-mesh sieve (particle size ≤ 250 μm), preferably through a 100-mesh sieve (particle size ≤ 150 μm), to obtain standardized dried powder of snow lotus cell culture.

[0079] In some embodiments, the obtained standardized snow lotus cell culture dried powder can also be subjected to standardized testing, including routine release tests and optional / periodic tests. The routine release tests are mandatory for each batch and include: source identification / verification of credential specimens (see step 110 for specific methods); microbial limit testing, performed according to General Chapter 1105 (microbial count method) and General Chapter 1106 (control bacteria test method) of the Chinese Pharmacopoeia; moisture determination, performed according to General Chapter 0832, Method II (drying method) of the Chinese Pharmacopoeia; and determination of characteristic component content, including total flavonoids, total polysaccharides, etc. In this embodiment, the characteristic component detection uses HPLC, and the specific detection steps are described in the following embodiments. Optional / periodic testing items are not required for every batch and can be performed according to quality standards and risk assessment results. These include: sterility testing (culture method), inoculating the test sample into a suitable culture medium and observing whether there is microbial growth; physicochemical index testing, such as ash content, pH, particle size, etc.; fingerprint spectroscopy detection, using HPLC or UPLC-HRMS; in the examples of this specification, the detection of yazolide is carried out using a broad-target metabolomics method (UHPLC-HRMS / MS), and the specific steps are described in the examples below.

[0080] Based on the above test results, batch release is determined as follows: if all indicators meet the standards, the batch is released into the warehouse, assigned a batch number, a sample is retained and archived, and the batch is put into the finished product warehouse or used for subsequent formulation development; if the batch does not meet the standards, it is not released, and a deviation investigation is initiated. Depending on the deviation, rework, downgrading, or scrapping is carried out, and re-inspection or termination of release may be necessary.

[0081] The aforementioned snow lotus cell culture and its use in the preparation of drugs for treating ovarian dysfunction will be described in detail below through several examples. It should be noted that the reaction conditions, reactants, and amounts of reactants in the examples are for illustrative purposes only and do not limit the scope of this specification.

[0082] Unless otherwise expressly stated, all percentages and contents in this instruction manual are by mass. Unless otherwise specified, all experimental methods used are conventional methods, and all materials and reagents used are commercially available. Furthermore, unless otherwise specified, SIC-L, SIC-M, and SIC-H represent the low, medium, and high dose groups of SIC, respectively.

[0083] Example 1: Preparation and quality characterization of standardized snow lotus cell cultures This embodiment provides a method for preparing a standardized snow lotus cell culture (hereinafter referred to as SIC) and a method for characterizing its quality. The SIC is derived from snow lotus (… Saussurea involucrata (Kar. et Kir.) Sch.-Bip.) The preparation process of dried material for in vitro cultured cells includes steps such as explant acquisition and sterilization, callus induction and subculture screening, suspension culture, scale-up, harvesting and drying, pulverization, and quality inspection. The following is a detailed description of each step.

[0084] 1. Acquisition and disinfection of explants.

[0085] The starting explants were selected from leaves, petioles, or root segments of *Saussurea involucrata*, as identified botanically; preferably, root segments, petioles, or leaves from sterile seedlings germinated from surface-sterilized seeds. Directly collected tissues were first washed with running water for 20 min, then treated with 70% ethanol aqueous solution for 30 s in a laminar flow hood, followed by treatment with 2% sodium hypochlorite aqueous solution for 10 min. If seeds were used as the starting material, the treatment was 70% ethanol for 30 s, followed by treatment with 2% sodium hypochlorite solution for 15 min. All treated materials were rinsed five times with sterile water, blotted dry with sterile filter paper, and cut into 2–3 mm pieces before inoculation. The laminar flow hood was wiped with 75% ethanol and irradiated with UV light for 30 min before operation, followed by turning off the UV light and ventilating for 10 min; both cutting and inoculation were performed using sterile forceps and a scalpel.

[0086] 2. Induction and subculture screening of callus tissue.

[0087] The explants were inoculated onto MS solid medium at pH 5.6–5.8 for callus induction. Root segments or petioles were inoculated onto a medium containing 0.3 mg / L 6-benzyladenine and 1.0 mg / L naphthaleneacetic acid (NAA); leaf explants were inoculated onto a medium containing 0.5 mg / L 6-benzyladenine and 1.5 mg / L NAA. Induction culture was conducted in the dark at 25 ± 2 °C. The obtained callus tissue was subcultured every 16–18 days, with callus fragments of 2–3 mm in diameter inoculated onto fresh medium. "Subculture" refers to the process of cutting the callus tissue and transferring it to fresh medium for passage culture to maintain its growth and differentiation capacity. During subculture, vigorous, compact, and non-water-soaked cell clusters were preferred; color was used only as an auxiliary observation indicator.

[0088] Proliferating cell lines were screened and subcultured for at least 10 generations. Using the last three generations of cultures as samples, the fold increase in fresh weight, total flavonoid content, and HPLC peak areas of chlorogenic acid, syringin, 1,5-O-dicaffeoylquinic acid, and rutin were determined, and the relative standard deviation (RSD) of each indicator was calculated. A stable cell line was defined as one where all RSDs did not exceed 15% and the chromatographic peaks of the four characteristic components were consistently detectable. The relative standard deviation is the ratio of the standard deviation of a set of data to its mean, used to measure batch-to-batch consistency. The HPLC detection methods for the four characteristic components are described in the quality characterization section below.

[0089] 3. Suspension culture.

[0090] The selected stable cell lines were inoculated into MS liquid medium to establish a suspension culture system. The MS liquid medium had the same hormone composition as the preferred root / petiole medium, containing 0.3 mg / L 6-benzyladenine and 1.0 mg / L naphthaleneacetic acid, but without added agar. The suspension culture conditions were as follows: inoculum size 20–30 g fresh cell weight / L, liquid volume in shake flasks 25% of the container volume, culture temperature 25±2 ℃, shaker speed 110±10 r / min, light conditions 12 h white light (1000–2000 lx) per day, and culture period 12–16 days.

[0091] Stable expanded cultures harvested should simultaneously meet the following conditions: cells exhibit regular morphology and dense cytoplasm under a microscope; ≥85% of cells are viable under trypan blue staining; fresh weight reaches 5–10 times the initial inoculum; the culture medium shows no abnormal turbidity, and smears are free of microorganisms under microscopic examination. The "trypan blue staining" is a commonly used method for detecting cell viability; viable cells, due to their intact cell membranes, reject trypan blue dye and are not stained blue.

[0092] 4. Gradually scale up the cultivation process.

[0093] The cultures obtained from suspension culture were scaled up stepwise in the order of shake flasks, seed tanks, intermediate tanks, and production tanks. The working volumes of each culture vessel were as follows: 0.15 L for shake flasks, 3 L or 5 L for seed tanks, 35 L or 50 L for intermediate tanks, and 350 L or 500 L for production tanks; the inoculum volume for each stage was 10% of the working volume of that stage. The culture temperature was 25±2 ℃, and the pH was controlled at 5.6–5.8; the stirring speeds for the seed tank, intermediate tank, and production tank were set to 80±5 r / min, 70±5 r / min, and 50–60 r / min, respectively; the aeration rate was 0.3–0.5 vvm, and dissolved oxygen was maintained at 20%–50% air saturation. "vvm" refers to the volume of gas introduced per minute per volume of culture medium, a commonly used unit for aeration. Samples were taken every 2 days to monitor temperature, pH, stirring speed, aeration rate, dissolved oxygen, cell morphology, viability, fresh weight, and sterility.

[0094] 5. Harvesting, drying and grinding.

[0095] The harvest endpoint is determined based on a combination of culture period, cell fresh weight, and cell viability: harvesting is considered complete when the culture period reaches 12–16 days, and two consecutive samples taken two days apart show a cell fresh weight increase of less than 10%, or cell viability decreases from an initial >90% to 85%–90%. The culture medium is removed by filtration or centrifugation, and the cells are quickly washed twice with purified water. The cells are then dried using methods such as freeze-drying, vacuum drying, or low-temperature drying not exceeding 60 °C to obtain a dried product. The dried product is then pulverized so that all of it passes through a 60-mesh sieve (particle size ≤250 μm), preferably a 100-mesh sieve (particle size ≤150 μm), to obtain SiC dry powder. Different drying and pulverization methods must not alter the detectability of the specified characteristic components.

[0096] 6. Identification of characteristic components by HPLC.

[0097] The HPLC test sample was prepared as follows: 0.5 g of SiC powder was added to 25 mL of 50%–75% methanol aqueous solution, and extracted by ultrasonication for 30 min. After cooling, the weight loss was adjusted, and the sample was centrifuged or filtered. The supernatant was then filtered through a 0.22 μm filter membrane. Chlorogenic acid, syringin, 1,5-O-dicaffeoylquinic acid, and rutin reference standards were prepared separately using the same solvent and then mixed.

[0098] Chromatographic analysis was performed using a reversed-phase C18 column with gradient elution of acidified aqueous phase and acetonitrile as the mobile phase. The detection wavelength was 265 nm. Column temperature, flow rate, and gradient can be adjusted according to the column, but the test sample and reference standard must be detected under the same conditions and in the same analytical sequence, and the peaks of the four reference standards should be distinguishably separated.

[0099] Figure 2This is an HPLC chromatogram for identifying characteristic components of standardized snow lotus cell cultures. Among them, Figure 2 The top image shows a mixed reference standard of chlorogenic acid, syringin, 1,5-O-dicaffeoylquinic acid, and rutin; the bottom image shows the SIC test sample. Figure 2 As shown, the retention times of chlorogenic acid, syringin, 1,5-O-dicaffeoylquinic acid, and rutin in the mixed reference standard were approximately 5.620 min, 10.444 min, 13.845 min, and 19.531 min, respectively; the SIC test sample showed corresponding peaks at approximately 5.621 min, 10.480 min, 13.971 min, and 19.633 min. The peaks were assigned according to the reference standards from the same batch, and the relative deviation of the retention times of each characteristic peak from the corresponding reference standard peak was less than 1.0%. The release limit is the detection of at least two of the characteristic components with a relative deviation of retention times not exceeding 2.0%.

[0100] 7. Detection of lactone chemical markers.

[0101] SIC uses yalianlactone as a marker for lactone quality. Lactone compounds are screened and annotated using a broad-target metabolomics method with UHPLC-ESI-QTRAP-MS / MS, and specificity is verified using certified reference standards or LC-HRMS / MS. Dehydroauristane lactone, lactucopicrin, ergosterol, and atractylodes lactone I are also annotated. Certified reference standards refer to reference materials prepared by authoritative institutions or accredited reference material manufacturers and accompanied by certificates, whose structure, purity, and characteristic values ​​have been rigorously verified. Broad-target metabolomics refers to a technical strategy that uses high-resolution mass spectrometry or triple quadrupole mass spectrometry to simultaneously detect and annotate as many known or unknown metabolites as possible in a sample.

[0102] The broad-target metabolomics test samples were prepared as follows: The same batch of SiC lyophilized samples were ground at 60 Hz for 60 s; 50 mg was accurately weighed and placed in a 2 mL centrifuge tube, and 700 μL of methanol-water extraction buffer (3:1, v / v) pre-cooled at -40 ℃ and containing an internal standard was added, followed by vortexing for 30 s; homogenization was carried out at 40 Hz for 4 min, followed by sonication in an ice-water bath for 5 min. The homogenization and sonication steps were repeated three times; extraction was carried out overnight at 4 ℃ with shaking. The extract was centrifuged at 12000 r / min for 15 min at 4 ℃, using a rotor radius of 8.6 cm, corresponding to a relative centrifugal force of approximately 13800 × g; the supernatant was filtered through a 0.22 μm microporous membrane, diluted 20-fold with the same extraction buffer, vortexed for 30 s, and then transferred to a sample vial. Equal volumes of 50 μL of each test sample were mixed to prepare QC samples; the test samples and QC samples were stored at -80 ℃ until analysis. QC samples are quality control samples used to monitor instrument stability and data quality during the analysis process.

[0103] High-performance liquid chromatography (HPLC) was performed using an ExionLC AD ultra-high performance liquid chromatography system and an ACQUITY UPLC HSS T3 column (2.1 mm × 100 mm, 1.8 μm). Mobile phase A was water containing 0.1% formic acid, and mobile phase B was acetonitrile. The column temperature was 40 °C, the autosampler temperature was 4 °C, the injection volume was 2 μL, and the flow rate was 0.40 mL / min. A feasible gradient program was as follows: 0–1.0 min, 5% B; 1.0–10.0 min, phase B linearly increased from 5% to 95%; 10.0–11.0 min, 95% B; 11.0–11.1 min, phase B decreased from 95% to 5%; 11.1–14.0 min, 5% B equilibration of the column. The gradient could be finely adjusted based on the column batch and system delay volume, but the test sample, QC sample, and reference standard should be detected under the same conditions.

[0104] Mass spectrometry was performed using a SCIEX QTRAP 6500+ triple quadrupole-linear ion trap mass spectrometer equipped with an IonDrive Turbo V electrospray ionization source, in multiple reaction monitoring (MRM) mode with switching between positive and negative ions. Multiple reaction monitoring refers to a technique where the mass spectrometer selects specific precursor ions for fragmentation and monitors their specific daughter ions to improve detection selectivity and sensitivity. Ion source parameters were: positive ion spray voltage +5500 V, negative ion spray voltage -4500 V, curtain gas 35 psi, ion source temperature 400 °C, ion source gas 1 60 psi, ion source gas 2 60 psi, and declustering voltage ±100 V. The precursor ions, quantitative daughter ions, qualitative daughter ions, and collision energies of each candidate lactone were optimized using corresponding certified reference standards. At least one pair of quantitative ions and one pair of qualitative ions were set for each compound, and solvent blanks, mixed QC, and the test sample were detected in the same analytical sequence.

[0105] Mass spectrometry data were acquired using SCIEX Analyst Work Station Software 1.6.3. Raw data were converted using MSConvert, and peak extraction, peak area integration, and candidate annotation were performed using a data processing program combined with a plant metabolite database. Broad-target results were first screened based on precursor ion-daughter ion pairs, retention times, and secondary fragment matching. When compounds listed in Table 1 are used as quality markers, they should further meet one of the following confirmation criteria: (a) co-analyzed with a certified reference standard under the same conditions, with a relative deviation in retention time not exceeding 2.0%, consistent qualitative ions, and a relative deviation in ion abundance ratio not exceeding 30%; or (b) verified using LC-HRMS / MS, with an excimer ion mass error not exceeding 5 ppm, and at least two diagnostic fragments consistent with the reference standard or a verified database record. Signals that do not meet the above specificity confirmation criteria are only recorded as candidate annotations and are not used alone for content determination or efficacy attribution.

[0106] Table 1. Routine Standard Information on Lactone Compounds Related to Standardized Snow Lotus Cell Cultures

[0107] To aid in characterizing the metabolic state and batch consistency of *Saussurea involucrata* cells during culture, live cell samples were collected at different stages of the culture process (including the callus stage, early suspension culture, logarithmic growth phase, and harvesting). Total RNA was extracted and reverse transcribed into cDNA. Using GAPDH2 as an internal control gene, the relative expression levels of related genes were detected using quantitative real-time PCR (qRT-PCR), and the 2^-ΔΔCt method was used for calculation. qRT-PCR is a method that uses real-time fluorescence quantitative polymerase chain reaction to monitor the PCR amplification process in real time, thereby quantifying specific nucleic acids in the starting template. An internal control gene is a gene that expresses relatively stably under different samples or conditions, used to correct for differences in RNA quantity, reverse transcription efficiency, and operational errors between samples. The 2^-ΔΔCt method is a commonly used relative quantification method based on comparing the cycle threshold (Ct), where ΔΔCt represents the difference in Ct values ​​between the target gene and the internal control gene under different treatments, and 2^-ΔΔCt reflects the fold change in the relative expression level of the target gene.

[0108] The genes detected included: HQT, HCT, FNS, UGT, OMT, CHS, F3H, and F3'H. Specifically, HQT is a gene related to hydroxycinnamoyl-CoA quinic acid transferase; HCT is a gene related to hydroxycinnamoyl-CoA shikimate / quinic acid transferase; FNS is a gene related to flavonoid synthase; UGT is a gene related to UDP-glycosyltransferase; OMT is a gene related to O-methyltransferase; CHS is a gene related to chalcone synthase; F3H is a gene related to flavanone 3-hydroxylase; and F3'H is a gene related to flavonoid 3'-hydroxylase. These genes mainly involve phenylpropanoid, flavonoid, and phenolic acid secondary metabolic pathways. The primer sequences of the plant genes used in this example are shown in Table 2.

[0109] Table 2. qRT-PCR primer sequences for genes related to the snow lotus cell culture process.

[0110] Figure 3 These are schematic diagrams showing the relative expression levels of HQT, HCT, FNS, UGT, OMT, CHS, F3H, and F3'H in callus tissue, suspension culture, scale-up culture, and harvest period, respectively. Figure 3 As shown, the aforementioned genes exhibited varying degrees of stage-specific expression changes during Saussurea involucrata cell culture. Compared to the callus stage, some genes among HQT, HCT, FNS, UGT, CHS, F3H, and F3'H showed higher relative expression levels in the early suspension culture, the logarithmic growth phase of scale-up culture, or the harvest period, suggesting that the transcriptional state related to phenolic acid, flavonoid, and phenylpropanoid metabolism can change with the culture stage. The expression patterns of different genes do not need to be completely consistent; these results are only used to assist in tracking metabolic status and batch consistency during culture and are not used as a separate release criterion for the dried product.

[0111] Example 2: Sesquiterpene lactone candidate genes, qRT-PCR validation protocol and sequence information This embodiment connects the characteristic lactone chemical features of Saussurea involucrata cell culture with its material basis for treating ovarian dysfunction. Specifically, candidate genes and their expression analysis are used to explain the biological basis for the possible formation of sesquiterpene lactone chemical features during the Saussurea involucrata cell culture stage; lactone compounds such as Saussurea lactone are used for the identification and batch consistency evaluation of dried SIC; Examples 3 to 8 verify the overall efficacy of the same standardized Saussurea involucrata cell culture (SIC) against cyclophosphamide-related ovarian dysfunction. The above three parts all point to the same traceable SIC material chain, without equating a single lactone component with the efficacy in an unverified causal relationship.

[0112] 1. Screening of candidate genes. Based on the obtained coding sequence (CDS), protein sequence, and transcriptome information related to costunolide from *Saussurea involucrata*, homology and expression association analyses were performed, resulting in the identification of six candidate genes associated with sesquiterpene lactone biosynthesis. Information on these candidate genes is shown in Table 3. Here, CDS refers to the coding sequence, i.e., the nucleotide sequence in a gene that directly encodes a protein. Costunolide is a sesquiterpene lactone compound, a common secondary metabolite found in plants of the Asteraceae family.

[0113] Table 3 Summary of candidate genes for sesquiterpene lactone synthesis in *Saussurea involucrata*

[0114] All qRT-PCR primers were designed for the provided CDS sequence, with the expected amplicon being 94–112 bp. Primer information is shown in Table 4.

[0115] Table 4. Primer information for qRT-PCR of candidate genes for sesquiterpene lactones from *Saussurea involucrata*.

[0116] To verify the expression characteristics of the aforementioned candidate genes during culture, qRT-PCR detection is recommended as follows: Cell samples should be collected at the callus stage, early suspension culture stage, logarithmic growth phase of scale-up culture, and harvest stage, with at least three independent biological replicates at each stage; each biological sample should be prepared in three technical replicates. Total RNA should be extracted from the samples and treated with DNase to remove genomic DNA contamination. DNase is a deoxyribonuclease used to digest residual DNA in the samples. Samples with an A260 / A280 ratio between 1.8 and 2.1 and meeting the RNA integrity requirements should be selected for reverse transcription to synthesize cDNA. A260 / A280 is the ratio of the absorbance of the nucleic acid sample at 260 nm and 280 nm wavelengths, used to assess nucleic acid purity.

[0117] Using GAPDH2 as an internal reference gene, real-time quantitative PCR (qRT-PCR) amplification was performed using the SYBR Green method. The "SYBR Green method" refers to a quantitative detection method that utilizes the fluorescence signal emitted by SYBR Green I fluorescent dye after binding to double-stranded DNA. The total reaction volume was 20 μL, including 10 μL of 2×SYBR Green premix, 0.4 μL each of forward and reverse primers (final concentration 0.2 μmol / L), 2 μL of diluted cDNA, and 7.2 μL of nuclease-free water. The recommended amplification program was: 95 ℃ pre-denaturation for 30 s; followed by 40 cycles: 95 ℃ denaturation for 5 s, 60 ℃ annealing and extension for 30 s; after cycling, melting curve analysis was performed at 65–95 ℃.

[0118] The acceptance criteria for amplification results are as follows: the melting curve shows a single peak; the amplified product shows a single band of the expected size on agarose gel electrophoresis; the linear regression coefficient R² of the standard curve is ≥0.99; the amplification efficiency is between 90% and 110%; and there is no specific amplification in either the template-free control or the reverse transcription-free control. The melting curve is a curve plotted based on the change in fluorescence signal after PCR amplification by gradually increasing the temperature to dissolve the double-stranded DNA; it is used to determine the specificity of the amplified product. The standard curve is a linear relationship curve between the Ct value and the logarithm of the template concentration obtained from amplification using serially diluted templates; it is used to calculate the amplification efficiency. The template-free control is a negative control without template, used to monitor reagent or operational contamination; the "reverse transcription-free control" is an RNA sample control that has not undergone reverse transcription, used to exclude interference from genomic DNA amplification.

[0119] Relative expression levels were calculated using the 2^-ΔΔCt method, where ΔΔCt represents the difference in the cycling threshold between the target gene and the internal reference gene in different samples, and 2^-ΔΔCt reflects the fold change in the relative expression level of the target gene. For data analysis, normality and homogeneity of variance were first tested, followed by one-way ANOVA and pre-defined multiple comparison methods for inter-group comparisons.

[0120] 3. Expression patterns of candidate genes Figure 5 This diagram illustrates the relative expression patterns of six candidate genes for sesquiterpene lactone biosynthesis in callus tissue, early suspension culture, logarithmic growth phase of scale-up culture, and live cells at harvest. The relative expression levels in the callus tissue stage are normalized to 1.0.

[0121] like Figure 5 As shown. The relative expression level in the callus stage was normalized to 1.0. Figure 5Figures A through F show the different stages of expression of six candidate genes for sesquiterpene lactone biosynthesis during snow lotus cell culture. Specifically, SiGAS1 (internal code SnowLotus37781) showed relative expression levels of approximately 2.2 and 4.0 in the early suspension culture and logarithmic growth phase of scale-up culture, respectively, and approximately 3.3 at harvest, indicating a moderate decline after reaching a high level during scale-up culture. The relative expression levels of SiGAS2 (SnowLotus27540) were approximately 1.0, 1.3, 2.0, and 1.7, showing a moderate overall increase. SiGAS-like1 (SnowLotus10635) showed little change in expression in the early culture stage, but rose to approximately 3.8 and 7.2 in the logarithmic growth phase of scale-up culture and at harvest, respectively. SiGAO1 (SnowLotus10621) gradually increased with the culture progress, with its relative expression level increasing from 1.0 to approximately 2.0, 3.1, and 4.4. SiGAO-like2 (SnowLotus12310) exhibited a non-monotonic, phased change, with relative expression levels of approximately 1.0, 1.5, 1.2, and 2.0, respectively. SiCOS-like1 (SnowLotus14648) showed a more significant increase in expression in the later stages of culture, gradually increasing from 1.0 in the callus stage to approximately 2.5, 5.9, and 8.6.

[0122] The above results indicate that different candidate genes have different stage-specific expression characteristics during the culture process, which can be used as auxiliary process indicators to characterize the metabolic state and batch consistency of snow lotus cell culture. However, their expression level is not used as a separate release standard for dried products.

[0123] 4. Functional annotation of candidate genes and inference of metabolic pathways Figure 4 This is a schematic diagram of the biosynthetic pathway and candidate genes of sesquiterpene lactones in Saussurea involucrata. Figure 4 The proposed metabolic pathway is illustrated, including the supply of mevalonate (MVA) precursors, the formation of farnesyl pyrophosphate (FPP), the formation of the germarene A skeleton, the oxidation of germarene A, and the formation of sesquiterpene lactone skeletons such as costone lactone. The “MVA pathway” is an important metabolic pathway for the synthesis of isoprene compounds in plants and animals; the “FPP” is farnesyl pyrophosphate, a key precursor in the biosynthesis of sesquiterpenes; and the “germarene A” is a sesquiterpene intermediate that can be further oxidized to generate various sesquiterpene lactones.

[0124] Combining sequence homology and Figure 5The staged expression pattern is shown, with SnowLotus37781 and SnowLotus27540 annotated as candidate genes for GAS, SnowLotus10621 as a candidate gene for GAO, and SnowLotus10635, SnowLotus12310, and SnowLotus14648 designated as SiGAS-like1, SiGAO-like2, and SiCOS-like1, respectively. GAS represents the gemmaene A synthase-related gene, GAO represents the gemmaene A oxidase-related gene, COS represents the costalactone lactone synthase-related gene, and "like" indicates a candidate homologous gene inferred based on sequence similarity but without functional verification. The gene function names are primarily annotated based on sequence homology, pathway location, and expression association; their specific catalytic functions can be further verified through heterologous expression, enzyme activity analysis, or genetic methods.

[0125] The internal instruction manual numbers for the CDS sequences, protein sequences, and primer sequences of the candidate genes used in this embodiment are shown in Table 5.

[0126] Table 5. Candidate Genes for Sesquiterpene Lactones (SEQ ID NO). Index

[0127] The candidate gene CDS sequences, protein sequences, qRT-PCR primer sequences, and expected amplification fragments involved in this embodiment are listed in SEQ ID NO: 1-24. For specific sequences, please refer to the sequence listing submitted with this specification.

[0128] Example 3: Cyclophosphamide-induced ovarian dysfunction model and treatment Eight-week-old female Wistar rats of SPF grade were selected and acclimatized under environmental conditions of 12 h / 12 ​​h light-dark alternation, temperature 20±2 ℃, and relative humidity 60±5%. "SPF grade" refers to specific pathogen-free experimental animals, meaning the animals do not carry specific pathogenic microorganisms that could interfere with the experiment. After the acclimatization period, the rats were randomly divided into a normal control group, a model group, an estradiol (E2) positive control group, a low-dose SIC group (SIC-L), a medium-dose SIC group (SIC-M), and a high-dose SIC group (SIC-H).

[0129] Except for the normal control group, the other groups received intraperitoneal injections of cyclophosphamide 50 mg / kg on day 1, and cyclophosphamide 8 mg / kg daily from day 2 to day 14 to induce chemotherapy-related ovarian dysfunction. The normal control group received an equal volume of normal saline at the same time.

[0130] After the modeling and drug administration were completed, continuous gavage treatment was administered for 21 days from day 15 to day 35. The normal control group and the model group were administered 10 mL / kg of distilled water by gavage daily; the E2 group was administered 0.18 mg / kg of estradiol by gavage daily; and the SIC-L, SIC-M, and SIC-H groups were administered SIC at 100 mg / kg, 210 mg / kg, and 420 mg / kg by gavage daily, respectively. Figure 6 Flowchart for cyclophosphamide modeling, SIC treatment, fertility monitoring, and sampling. See the experimental procedure below. Figure 6 .

[0131] Sampling was completed on day 36. Serum and ovarian tissue were collected, and body weight and ovarian wet weight were recorded; some animals were used for subsequent fertility observation. The efficacy conclusions described in this example are based on a comprehensive analysis of serum hormone levels, ovarian histology, follicle count, ovarian gene expression, and the detection results of sterols or steroid hormone-related metabolites.

[0132] Example 4: Ovarian endocrine function After drug administration, serum was separated, and the levels of anti-Müllerian hormone (AMH), estradiol (E2), and follicle-stimulating hormone (FSH) were measured using a blinded coded sample method. Blinding means that the operator is unaware of the sample group during the testing process to reduce human bias. The tests were performed using the Elabscience rat AMH (E-EL-R3022), QuicKey Pro rat E2 (E-OSEL-R0001), and rat FSH (E-EL-R0391) ELISA kits, or other kits with equivalent performance and validated with rat serum. ELISA is an enzyme-linked immunosorbent assay that uses antigen-antibody specific reactions and enzyme-catalyzed substrate color development to quantitatively detect the concentration of target proteins in a sample. A standard curve was established according to the kit instructions, and each sample was tested in replicates. Repeat testing was performed when the coefficient of variation (CV) of the replicates was greater than 15%. The coefficient of variation, the ratio of the standard deviation to the mean, is used to measure the consistency of repeated measurements.

[0133] The results are as follows Figure 7 As shown. Figure 7 This is a schematic diagram illustrating the effects of SIC on serum E2, AMH, and FSH in rats, as shown in some examples of this specification. Figure 7In this document, "control" represents the normal control group, and "model" represents the model group. The normal control group refers to the experimental animal group that did not receive any modeling treatment but was given the same solvent or blank treatment as the treatment group. In the examples of this specification, the normal control group rats were intraperitoneally injected with an equal volume of physiological saline at the same time points and were administered distilled water by gavage daily during the treatment period. This group is used to reflect the baseline levels of various test indicators under normal physiological conditions, serving as a reference for evaluating the degree of ovarian function damage and the effect of drug intervention. The model group refers to the experimental animal group that received modeling treatment but did not receive active drug intervention, only solvent or blank treatment. In the examples of this specification, the model group rats were induced with chemotherapy-related ovarian function damage by intraperitoneal injection of cyclophosphamide (50 mg / kg on day 1, 8 mg / kg daily from day 2 to 14), and were administered distilled water by gavage daily after the modeling was completed. This group is used to simulate the pathological state of ovarian function damage after chemotherapy, serving as a negative control for evaluating the therapeutic effect of SIC. The E2 group refers to the experimental animal group that received estradiol (E2) as a positive control drug. In the examples described in this specification, rats in the E2 group were administered estradiol 0.18 mg / kg daily by gavage for 21 consecutive days after the cyclophosphamide model was established. Estradiol is the main estrogen secreted by the ovaries, and exogenous estradiol supplementation can improve hormonal imbalances and target organ function related to ovarian dysfunction to some extent. Therefore, this group was used to verify the model's responsiveness to estrogen replacement therapy and as a reference for evaluating the effect of SIC in improving ovarian endocrine function. L, SIC M and SIC Group H refers to the groups treated with low, medium, and high doses of the test drug, namely, standardized snow lotus cell culture (SIC). In the embodiments of this application, after the cyclophosphamide model was established, the rats in the above groups were administered SIC at doses of 100 mg / kg (low dose), 210 mg / kg (medium dose), and 420 mg / kg (high dose) by gavage daily for 21 consecutive days. The purpose of setting up different dose groups was to observe the dose-response relationship of SIC on chemotherapy-related ovarian dysfunction and to determine the dose range that can produce a stable therapeutic effect. Figure 7 in, # and The significance marker is #, indicating a comparison with the normal control group. The significance level is indicated by the number of signs compared to the model group. All data are expressed as mean ± standard deviation. One-way ANOVA and multiple comparisons were used for comparisons between groups. The significance level is indicated by the number of signs. For example, ### indicates that the group is compared with the normal control group by P < 0.001; , and These represent P<0.05, P<0.01, and P<0.001 respectively when comparing this group with the model group. P refers to the probability value in statistics. The smaller the P value, the lower the probability that the difference between the two groups of data is caused by random error, and the more likely the difference is to be real.

[0134] The model group showed significantly lower E2 and AMH levels and significantly higher FSH levels compared to the normal control group, indicating that cyclophosphamide induced damage to ovarian reserve and endocrine function. Compared to the model group, SIC-L, SIC-M, and SIC-H all increased E2 and AMH levels; SIC-M and SIC-H significantly decreased FSH levels. The SIC-H group showed E2 and AMH levels close to those of the E2-positive control group. These results suggest that SIC administration after modeling can still improve ovarian endocrine function, with medium and high doses showing more stable effects than lower doses.

[0135] Example 5: Ovarian Histology and Follicle Count Ovarian tissue was fixed in 4% paraformaldehyde solution for 24 h, followed by graded ethanol dehydration and paraffin embedding to prepare 5 μm serial sections. Paraffin embedding involves immersing the fixed, dehydrated tissue in molten paraffin, allowing it to cool and form a hard block for thin slicing. One section every five slides was selected for hematoxylin-eosin (HE) staining. HE staining is a commonly used histological staining method; hematoxylin makes the cell nuclei appear blue, while eosin makes the cytoplasm and extracellular matrix appear red, facilitating observation of tissue structure. Under a blinded grouping method, the number of primordial follicles, primary follicles, secondary follicles, and atretic follicles was counted.

[0136] Figure 8A HE staining images of the normal control group, model group, E2 group, SIC-L group, SIC-M group and SIC-H group, in order. Figure 8A The figures in the middle reflect the effects of SIC on ovarian tissue morphology and follicle count in the normal control group, model group, E2 group, SIC-L group, SIC-M group and SIC-H group, respectively. Figure 8B The diagram shows the counting of atretic follicles, secondary follicles, primordial follicles, and primary follicles, in that order.

[0137] HE staining (Hematoxylin) Eosin staining, i.e., hematoxylin Eosin staining is a staining method used in histopathological sections. HE staining utilizes the different affinities of two dyes for different cell structures to achieve a clear distinction between the cell nucleus and cytoplasm, as well as different tissue components.

[0138] like Figure 8AAs shown in the figure, the ovarian structure of the normal control group was intact, and follicles at different developmental stages were visible; the ovarian structure of the model group was damaged and the number of atretic follicles increased; the ovarian morphology of the E2 group and the SIC treatment group showed varying degrees of recovery.

[0139] Figure 8B In the figure, ### indicates that compared with the normal control group, P<0.001; This indicates that the p-value for this group is less than 0.001 compared to the model group. For example... Figure 8B As shown, the model group exhibited an increase in atretic follicles and a decrease in primordial, primary, and secondary follicles. Compared to the model group, SIC-M and SIC-H significantly reduced atretic follicles and increased primordial, primary, and secondary follicles; SIC-L showed a more significant improvement in primary follicles. Histological and follicle count results were consistent with serum endocrine results, indicating that SIC not only alters serum hormone levels but also improves ovarian tissue structure and follicular reserve.

[0140] Example 6: Genes related to ovarian function and steroid production Figure 9 This diagram illustrates the effect of SIC on the relative expression levels of Cyp17a1, Cyp19a1, Hsd17b1, Star, Scarb1, and Amh in ovarian tissue. Ovarian tissue was collected from the normal control group, model group, and SIC-H group (420 mg / kg body weight / day), and the relative expression levels of Cyp17a1, Cyp19a1, Hsd17b1, Star, Scarb1, and Amh genes were detected. The results are shown below. Figure 9 Among them, Cyp17a1 encodes member 1 of subfamily A of cytochrome P450 family 17, which participates in 17α-hydroxylation and 17,20-cleavage reactions; Cyp19a1 encodes aromatase, which catalyzes the conversion of androgens to estrogens; Hsd17b1 encodes 17β-hydroxysteroid dehydrogenase 1, which participates in steroid hormone metabolism; Star encodes acute regulatory protein of steroid production, which is responsible for the transport of cholesterol to the inner mitochondrial membrane; Scarb1 encodes member 1 of scavenger receptor class B, which participates in cholesterol uptake; and Amh encodes anti-Müllerian hormone, which is associated with ovarian reserve and follicle development. Figure 9 In the figures, #, ##, and ### represent P<0.05, P<0.01, and P<0.001 respectively compared with the normal control group. , and The values ​​represent P<0.05, P<0.01, and P<0.001, respectively, when compared to the model group.

[0141] For each sample, 50–100 mg of ovarian tissue was used, and total RNA was extracted using the TRIzol method. The TRIzol method is a standard method for cell lysis and RNA separation using a lysis buffer containing phenol and guanidine isothiocyanate. The extracted total RNA was tested for purity and integrity; samples with an A260 / A280 ratio between 1.8 and 2.0 and meeting the RNA integrity requirements were used for subsequent experiments. 1 μg of total RNA was reverse transcribed into cDNA, and real-time quantitative PCR (qRT-PCR) was performed using the SYBR Green method with the cDNA as a template. The total reaction volume was 20 μL, containing 2 μL of cDNA, 0.4 μmol / L final concentrations of upstream and downstream primers, 10 μL of 2×SYBR Green MasterMix, and nuclease-free water. Amplification conditions were: 95 ℃ pre-denaturation for 3 min; followed by 40 cycles of 95 ℃ for 15 s, 60 ℃ for 30 s, and 72 ℃ for 30 s. Using Gapdh as an internal reference gene, the relative expression level was calculated using the 2^-ΔΔCt method; primer sequences are shown in Table 6.

[0142] Table 6 Primer sequences for qRT-PCR of rat ovarian tissue

[0143] like Figure 9 As shown, compared with the normal control group, the relative expression levels of Cyp17a1, Cyp19a1, Hsd17b1, and Amh genes decreased in the model group, while the relative expression levels of Star and Scarb1 genes increased. After SIC treatment, the expression levels of Cyp17a1, Cyp19a1, Hsd17b1, and Amh were significantly upregulated, while the abnormal increases in Star and Scarb1 were suppressed.

[0144] Of the aforementioned genes, Cyp17a1, Cyp19a1, and Hsd17b1 encode key enzymes involved in the steroid hormone synthesis pathway, closely related to the production of androgens and estrogens; Amh encodes anti-Müllerian hormone, associated with follicular granulosa cell function and ovarian reserve; Star and Scarb1 encode an acute regulatory protein for steroid production and a scavenger receptor class B member 1, respectively, involved in cholesterol transport to mitochondria and substrate supply. These results indicate that SIC can correct the imbalance of the steroid production network under ovarian injury, but this should not be interpreted as SIC having a direct binding or direct regulatory effect on any single gene or protein; the changes described are merely accompanying molecular events in overall pharmacological efficacy.

[0145] Example 7: Sterols and Steroid Hormone-Related Metabolites Ovarian tissue samples were collected from the normal control group, model group, and SIC-H group (420 mg / kg body weight / day). Sterols and steroid hormone-related metabolites were detected, and the relative levels of pregnenolone, desmosterol, 7-ketocholesterol, 7α,25-dihydroxycholesterol, 25-hydroxycholesterol, corticosterone, testosterone, progesterone, androstenedione, 17α-hydroxyprogesterone, and 17β-estradiol were compared. Sterols are alcohol compounds with a cyclopentane-polyhydrophenanthrene core; cholesterol and its oxidized derivatives belong to this category. Steroid hormones are hormones synthesized from cholesterol precursors, including estrogens, androgens, progestins, and corticosteroids. Detection results are shown in [link to results]. Figures 10A-10B In the figure, SIC refers to group SIC-H. Figures 10A-10B These are schematic diagrams illustrating the effects of SIC on pregnenolone, desmosterol, 7-ketocholesterol, 7α,25-dihydroxycholesterol, 25-hydroxycholesterol, corticosterone, testosterone, progesterone, androstenedione, 17α-hydroxyprogesterone, and 17β-estradiol. Figures 10A-10B In the diagram, significant differences between groups are marked with an asterisk next to the corresponding data points, for example, This indicates that P < 0.05. This indicates that P < 0.01. This indicates that P < 0.001. This indicates that P < 0.0001. See [link to comparison details] for more information. Figures 10A-10B Connecting the broken lines.

[0146] Compared with the normal control group, the relative levels of pregnenolone, 7-ketocholesterol, 7α,25-dihydroxycholesterol, 25-hydroxycholesterol, and corticosterone were increased in the model group, while the relative levels of sterols, testosterone, progesterone, androstenedione, 17α-hydroxyprogesterone, and 17β-estradiol were decreased. After SIC treatment, the elevated levels of metabolites in the model group decreased, and the decreased levels of metabolites increased. All 11 metabolites tested showed a trend of reverting to the normal control level, and the inter-group comparisons shown in the figure reached statistical significance.

[0147] The above changes in metabolites and Figure 9 The gene expression changes shown and Figure 7 The results of serum E2 recovery corroborate each other, jointly supporting the association between SIC and the restoration of cholesterol conversion and steroid hormone synthesis homeostasis. It should be noted that the existing data are inter-group comparisons and should not be construed as establishing a causal relationship between a single component and drug efficacy.

[0148] Example 8: Dosage-Efficacy Relationship Analysis of serum hormone levels and follicle count results showed that the SIC 100 mg / kg dose group had an improving effect on E2, AMH levels and the number of primary follicles; the 210 mg / kg and 420 mg / kg dose groups showed more consistent and stable improvement effects on E2, AMH, FSH levels and atretic follicles, primordial follicles, primary follicles and secondary follicles.

[0149] Therefore, in the animal models used in the embodiments of this specification, 100–420 mg / kg body weight / day is the effective oral dose range, of which 210–420 mg / kg body weight / day is the preferred dose range. This dose range is used to support animal efficacy experiments and pharmaceutical applications and is not directly equivalent to the human clinical use dose.

[0150] Example 9: Preparation of enriched fractions and specific oral formulation This embodiment provides a method for preparing enriched fractions of snow lotus cell culture and an exemplary formulation and process parameters for an oral preparation containing SiC.

[0151] 1. Preparation of enrichment sites Take 100 g of dried SiC powder and add 1000 mL of 50% (v / v) ethanol aqueous solution. Reflux extraction is performed twice at 80±2 ℃, 1 h each time. Reflux extraction refers to heating the extraction solvent to boiling, condensing the solvent vapor, and then refluxing it back into the extraction container to extract the medicinal material in a circulating manner. After each extraction, filter while hot and combine the filtrates. Recover the ethanol from the combined filtrate under reduced pressure at no more than 60 ℃ and -0.08 to -0.06 MPa, and concentrate to a relative density of 1.10–1.20 (measured at 60 ℃). Relative density is the ratio of the density of a substance at a certain temperature to the density of a reference substance; here, it is used to control the concentration of the concentrate. Load the concentrate onto a D101 macroporous adsorption resin column at a flow rate not exceeding 1 column volume / hour. Macroporous adsorption resin is a polymeric adsorbent with a macroporous structure that can separate components in a mixture through hydrophobic interactions, hydrogen bonds, etc. After loading the sample, it was first eluted with 3 column volumes of purified water to remove impurities, followed by 3 column volumes of 30% ethanol aqueous solution. The 30% ethanol eluent was collected. The eluent was concentrated under reduced pressure at a temperature not exceeding 60 °C and then freeze-dried to obtain the enriched fraction. Freeze-drying is a drying method that removes moisture by directly sublimating ice in frozen materials into water vapor under low temperature and reduced pressure conditions, which is beneficial for maintaining the stability of the material composition.

[0152] The obtained enriched fraction was identified using the HPLC method described in Example 1, based on the peaks of 1,5-O-dicaffeoylquinic acid and rutin reference standards from the same batch. A qualified enriched fraction should simultaneously detect the corresponding chromatographic peaks of the above two components, and the relative deviation of the retention times should not exceed 2.0%. This enriched fraction is currently disclosed only as a preparation method and material for subsequent research; since the examples in this specification do not provide independent animal efficacy test results, the efficacy results of the dried SiC product are not directly equivalent to those of this enriched fraction, nor is any therapeutic use of the enriched fraction claimed based on this.

[0153] 2. Oral formulation prescription To demonstrate the feasibility of pharmaceutical compositions containing SIC, this embodiment provides specific exemplary formulations and key process parameters for tablets, capsules, granules, pills, powders, oral solutions, suspensions, and lyophilized powders reconstituted before oral administration, as shown in Table 7. Each formulation is exemplified by containing 100 mg of SIC per unit; during clinical development, the unit dose can be adjusted based on dosage conversion and formulation validation results, for example, to contain 50–500 mg of SIC per unit.

[0154] Table 7 Formulation and Key Process Parameters of SIC Oral Formulations

[0155] The excipients for each dosage form listed in Table 7 should meet pharmaceutical standards. During preparation, the amount of SiC added, mixing uniformity, moisture content, fill weight variation, and microbial limits should be controlled. The HPLC method described in Example 1 should be used to confirm that the characteristic components in the formulation are detectable and that peak assignment is not affected by excipients. All labeled amounts for each formulation are calculated based on dried SiC. Formulations can be equivalently adjusted without changing the identity of the active ingredient, unit dosage, and quality control requirements.

[0156] Unlike wild snow lotus raw material or general extracts, the embodiments in this specification explicitly use in vitro expanded snow lotus cell cultures as the object, and characterize the same material using the expression of sesquiterpene lactone candidate genes during the culture process, HPLC characteristic components, and lactone chemical markers represented by snow lotus lactone. This standardized material was subsequently used for efficacy evaluation of cyclophosphamide-related ovarian dysfunction, enabling a natural connection between the chemical quality attributes of the material and the indication for ovarian dysfunction through the "traceable quality attributes of the same SIC," avoiding the problem of unstable or non-reproducible efficacy evaluation results due to differences in raw material source and batch.

[0157] After cyclophosphamide modeling was completed, oral administration for 21 consecutive days resulted in the improvement of serum estradiol (E2) and anti-Müllerian hormone (AMH) levels in rats with ovarian dysfunction. Medium- and high-dose groups also significantly reduced follicle-stimulating hormone (FSH) levels, and these effects increased with increasing dose. This indicates that SIC has a clear therapeutic effect on ovarian endocrine dysfunction caused by chemotherapy, rather than merely a preventative effect.

[0158] Regarding ovarian tissue morphology, SIC can improve ovarian structural damage caused by chemotherapy. In the medium- and high-dose groups, the number of atretic follicles decreased, while the number of primordial, primary, and secondary follicles increased, indicating that SIC has a protective effect on follicles at different developmental stages in the ovary and helps maintain or restore ovarian follicular reserve.

[0159] Furthermore, after SIC administration, changes in the expression of ovarian function and steroid production-related genes (such as Cyp17a1, Cyp19a1, Hsd17b1, Amh, Star, Scarb1, etc.) as well as changes in the levels of 11 sterols or steroid hormone-related metabolites all reverted to the normal control trend. These molecular-level changes, along with improvements in serum hormone levels and ovarian histology, corroborate each other, supporting the therapeutic effect of SIC by restoring steroid production network homeostasis and improving ovarian reserve and endocrine function.

[0160] The basic concepts have been described above. Obviously, for those skilled in the art, the detailed disclosure above is merely illustrative and does not constitute a limitation of this specification. Although not explicitly stated herein, those skilled in the art may make various modifications, improvements, and corrections to this specification. Such modifications, improvements, and corrections are suggested in this specification and therefore remain within the spirit and scope of the exemplary embodiments described herein.

[0161] Furthermore, this specification uses specific terms to describe embodiments thereof. For example, "an embodiment," "one embodiment," and / or "some embodiments" refer to a particular feature, structure, or characteristic associated with at least one embodiment of this specification. Therefore, it should be emphasized and noted that references to "an embodiment," "one embodiment," or "an alternative embodiment" in different locations throughout this specification do not necessarily refer to the same embodiment. Moreover, certain features, structures, or characteristics in one or more embodiments of this specification can be appropriately combined.

[0162] Similarly, it should be noted that, in order to simplify the description disclosed herein and thus aid in the understanding of one or more embodiments of the invention, the foregoing description of embodiments in this specification may sometimes combine multiple features into a single embodiment, drawing, or description thereof. However, this method of disclosure does not imply that the subject matter of this specification requires more features than those mentioned in the claims. In fact, the embodiments contain fewer features than all the features of a single embodiment disclosed above.

[0163] In some embodiments, numbers describing the quantity of components and attributes are used. It should be understood that such numbers used in the description of embodiments are modified in some examples with the terms "approximately," "approximately," or "generally." Unless otherwise stated, "approximately," "approximately," or "generally" indicates that the numbers are allowed to vary by ±20%. Accordingly, in some embodiments, the numerical parameters used in the specification and claims are approximate values, which may be changed depending on the characteristics required by individual embodiments. In some embodiments, numerical parameters should take into account specified significant digits and employ a general method of digit reservation. Although the numerical ranges and parameters used to confirm their breadth of range in some embodiments of this specification are approximate values, in specific embodiments, such values ​​are set as precisely as feasible.

[0164] Finally, it should be understood that the embodiments described in this specification are merely illustrative of the principles of the embodiments described herein. Other variations may also fall within the scope of this specification. Therefore, alternative configurations of the embodiments described herein are intended to be illustrative rather than limiting, and should be considered consistent with the teachings of this specification. Accordingly, the embodiments described herein are not limited to those explicitly introduced and described herein.

Claims

1. The use of a standardized snow lotus cell culture in the preparation of a drug for treating chemotherapy-related ovarian dysfunction, characterized in that, The standardized snow lotus cell culture is a dried cell culture obtained by expanding, harvesting, and drying in vitro cultured cells of the genus Snow Lotus; the dried cell culture contains at least two of the following: chlorogenic acid, syringin, 1,5-O-dicaffeoylquinic acid, and rutin.

2. The use according to claim 1, characterized in that, The plant in question is Saussurea involucrata (Kar. et Kir.) Sch.-Bip.; the dried cell culture contains chlorogenic acid, syringin, 1,5-O-dicaffeoylquinic acid and rutin.

3. The use according to claim 1 or 2, characterized in that, The chemotherapy-related ovarian dysfunction is induced by cyclophosphamide or its active metabolites; the drug is administered after cyclophosphamide administration has ended and ovarian damage has already occurred.

4. The use according to claim 3, characterized in that, The therapeutic effects of the drug include: compared with an untreated chemotherapy-related ovarian dysfunction model, one or more of the following in the serum of the subjects after administration: increased levels of anti-Müllerian hormone (AMH), increased levels of estradiol (E2), and decreased levels of follicle-stimulating hormone (FSH).

5. The use according to claim 3, characterized in that, The therapeutic effects of the drug also include: an increase in the absolute number of primordial, primary, or secondary follicles in ovarian tissue sections of subjects after administration, and / or a decrease in the proportion of atretic follicles to the total number of follicles, compared with an untreated chemotherapy-related ovarian dysfunction model.

6. The use according to claim 3, characterized in that, The therapeutic effects of the drug also include changes in the ovarian tissue of subjects after administration, compared to an untreated model of chemotherapy-related ovarian dysfunction, including at least one of the following: Upregulation of mRNA expression levels of Cyp17a1, Cyp19a1, Hsd17b1 or anti-Müllerian hormone; Decreased mRNA expression levels of acute regulatory steroid protein (Star) or scavenger receptor class B member 1 (Scarb1); or, Pregnenolone, desmosterol, 7-ketocholesterol, 7α,25-dihydroxycholesterol, 25-hydroxycholesterol, corticosterone, testosterone, progesterone, androstenedione, 17α-hydroxyprogesterone, or 17β-estradiol return to normal physiological levels.

7. The use according to any one of claims 1 to 6, characterized in that, The drug is an oral preparation selected from tablets, capsules, granules, pills, powders, oral liquids, suspensions, and lyophilized powders reconstituted before oral administration; the unit dose of the preparation contains 50-500 mg of the dried cell culture.

8. A pharmaceutical composition, characterized in that, It comprises a standardized snow lotus cell culture and pharmaceutically acceptable excipients; the standardized snow lotus cell culture is a dried cell culture obtained by expanding, harvesting and drying in vitro cultured cells of snow lotus (Saussurea involucrata (Kar. et Kir.) Sch.-Bip.); the dried cell culture contains at least two of chlorogenic acid, syringin, 1,5-O-dicaffeoylquinic acid and rutin.

9. The pharmaceutical composition according to claim 8, characterized in that, The pharmaceutical composition is used to treat chemotherapy-related ovarian dysfunction induced by cyclophosphamide or its active metabolites; the pharmaceutical composition is in the form of tablets, capsules, granules, pills, powders, oral liquids, suspensions, or lyophilized powder reconstituted before oral administration.

10. A method for preparing a standardized snow lotus cell culture, characterized in that, include: To obtain sterile explants or callus tissue from plants of the genus Saussurea; Establish and expand in vitro cultured cells; Harvest cells and remove any remaining culture medium; Drying and pulverizing yields dried cell culture material; The dried cell culture contains at least two of chlorogenic acid, syringin, 1,5-O-dicaffeoylquinic acid, and rutin.

11. The preparation method according to claim 10, characterized in that, The establishment and expansion of in vitro cultured cells includes: Inoculate 20–30 g of fresh cell weight per liter of culture medium and culture for 12–16 days at 23–27 °C and 100–120 r / min. Scale up the cells through at least two stages of culture in shake flasks, seed tanks, and production tanks.

12. A quality control method for standardized snow lotus cell cultures, characterized in that, include: High performance liquid chromatography was used to compare the retention times of each characteristic peak in the chromatogram of the test sample with those of the reference standards chlorogenic acid, syringin, 1,5-O-dicaffeoylquinic acid and rutin at a detection wavelength of 265 nm. The structure of yazolide was confirmed using LC-HRMS / MS. Batches containing at least two of chlorogenic acid, syringin, 1,5-O-dicaffeoylquinic acid, and rutin, with the relative deviation of the retention time of each characteristic peak from the corresponding reference peak not exceeding 2.0%, are deemed to meet the requirements for characteristic component identification.

13. The quality control method according to claim 12, characterized in that, The method further includes process control of live cell samples before harvesting, the process control including: Using GAPDH2 as an internal reference gene, qRT-PCR was used to detect the relative expression levels of at least one of the following genes: hydroxycinnamoyl-CoA quinoline hydroxycinnamoyltransferase (HQT), shikimate O-hydroxycinnamoyltransferase (HCT), flavonoid synthase (FNS), UDP-glycosyltransferase (UGT), O-methyltransferase (OMT), chalcone synthase (CHS), flavanone 3-hydroxylase (F3H), and flavonoid 3'-hydroxylase (F3'H), as well as at least two of the sesquiterpene lactone candidate genes with sequences as shown in SEQ ID NO: 1–24. This was used to help characterize the culture stage and batch consistency.

14. A method for preparing enriched fractions from snow lotus cell cultures, characterized in that, Includes the following steps: Snow lotus ( Saussurea involucrata (Kar. et Kir.) Sch.-Bip.) The dried product of in vitro cultured cells was extracted twice by reflux at 80±2 °C for 1 h each time, with 10 mL of 50% ethanol aqueous solution added per gram of dried product. The filtrates were then combined. The combined filtrates were concentrated under reduced pressure at ≤60 ℃ and -0.08~-0.06 MPa to a relative density of 1.10~1.20 (measured at 60℃) to obtain a concentrated solution; The concentrate was loaded onto a D101 macroporous adsorption resin column at a flow rate of no more than 1 column volume / hour. It was first eluted with 3 column volumes of purified water to remove impurities, and then eluted with 3 column volumes of 30% ethanol aqueous solution. The 30% ethanol eluent was collected. The 30% ethanol eluent was concentrated under reduced pressure at ≤60 °C and then freeze-dried to obtain an enriched fraction; the enriched fraction contained 1,5-O-dicaffeoylquinic acid and rutin.

Images