Lithospermum plant extracts with high acetylshikonin content, compositions for imparting anti-influenza activity, foods and beverages utilizing Lithospermum plants, clothing utilizing Lithospermum plants, miscellaneous goods utilizing Lithospermum plants, and products utilizing Lithospermum plants.

Abstract

To develope a novel and efficient extraction method for acetylshikonin and shikonin, which are the active ingredients of Lithospermum erythrorhizon to contribute to the effective use of Lithospermum erythrorhizon.SOLUTION: The present invention is a Lithospermum plant extract for imparting anti-influenza effect and the anti-influenza effect includes both preventive effect of influenza and treatment effect of influenza. Ethanol, more preferably ethanol to which d-limonene is added, can be used to extract the Lithospermum plant extract of the invention. As raw materials, either of Lithospermum erythrorhizon Siebold et Zucc., Lithospermum officinale L., Lithospermum arvense L., or Lithospermum zollingeri A.DC. can be suitably used.SELECTED DRAWING: None

Description

Technical Field

[0001] The present invention High-acetylshikonin-containing Lithospermum plant extract, composition for imparting anti-influenza activity, food and beverages utilizing Lithospermum plants. relates to the following, etc., and particularly relates to an anti-influenza virus technology using a plant of the genus Viola as a raw material.

Background Art

[0002] Conventionally, various research and developments have been carried out on plant-derived antiviral agents and the like, and patent applications have also been made. For example, Patent Document 1 cited below discloses an anti-influenza virus agent containing a proanthocyanidin-containing plant extract, a propolis extract, and lactic acid bacteria. As the proanthocyanidin-containing plant, berries such as cranberry are particularly mentioned, and ethanol is used for extracting its extract.

[0003] Further, Patent Document 2 discloses an antiviral agent containing, as an active ingredient, a plant-derived extract having an α-glucosidase inhibitory action, and examples of the plant-derived extract include a water extract or an alcohol extract of a plant of the genus Salacia. It is described that it is particularly for preventing influenza virus infection and reducing its symptoms.

[0004] Further, Patent Document 3 discloses a supernatant obtained by grinding the edible part of yam and centrifuging it at 10,000 rpm or less for 1 hour or less as an orally ingestible anti-influenza virus composition. Furthermore, it is described that the viscosity can be reduced by appropriately subjecting this to ultrasonic treatment, and it can be dried and powdered by freeze-drying or the like.

Prior Art Documents

Patent Documents

[0005]

Patent Document 1

Patent Document 2

[0006] Thus, various antiviral agents and antiviral compositions derived from plant resources have been proposed. Lithospermum erythrorhizon Siebold et Zucc., a member of the Boraginaceae family, has also had its roots (purple gromwell) used as a dye and as a pharmaceutical product exhibiting anti-inflammatory and antitumor effects (see Reference 1 below). Shikonin, a component of purple gromwell, has antiviral effects against influenza A virus (Reference 2), hepatitis C virus (Reference 3), adenovirus (Reference 4), enterovirus (Reference 5), and HIV (Reference 6), and acetylshikonin, a shikoin derivative, also has anticoxsackievirus effects (Reference 7), so its effective utilization is promising.

[0007] However, regarding the purple gromwell, cultivation techniques are not yet established, and its population is declining due to environmental changes caused by global warming, despite its suitability for cool climates (as of March 2022, it is listed as Endangered 1B on the Ministry of the Environment's Red List (Ministry of the Environment Red List 2020)). Therefore, further advancement of research and development focused on its effective utilization is awaited. Under these circumstances, it is important to continue research that can further expand the possibilities for the effective utilization of purple gromwell, in order to promote the efforts of the Kami Town Medicinal Plant Research Association (established in November 2015) in Kami Town, Kami District, Miyagi Prefecture, to improve its cultivation techniques.

[0008] Therefore, the objective of this invention is to develop a novel and efficient extraction method for acetylshikonin and shikonin, which are active ingredients of Lithospermum erythrorhizon, based on the existing technologies and efforts, and to contribute to the effective utilization of Lithospermum erythrorhizon. [Means for solving the problem]

[0009] The inventors of this application have investigated the above-mentioned problems. As a result, they have developed a novel extraction method for extracting acetylshikonin and shikonin, which are active ingredients of Lithospermum erythrorhizon, at high concentrations, and have confirmed that Lithospermum erythrorhizon ethanol extract has an influenza virus inactivating effect, and that this effect is further enhanced in d-limonene-added ethanol extract, which can efficiently extract acetylshikonin at high concentrations. Based on these findings, the inventors have completed the present invention. That is, the invention claimed in this application, or at least the invention disclosed, as a means of solving the above-mentioned problems, is as follows.

[0010] [1] The extraction is performed using ethanol (95) to which d-limonene has been added, characterized in that the amount of d-limonene added to the ethanol is 5% by volume or more. For conferring anti-influenza activity High acetylshikonin content Extract from plants of the Lithospermum genus. [2] Ethanol (95) to which d-limonene has been added is used for extraction, and the amount of d-limonene added to the ethanol is 5% by volume or more. The aforementioned anti-influenza effect is characterized by being an influenza preventive effect, as described in [1]. High acetylshikonin content Extract from plants of the Lithospermum genus. [3] Ethanol (95) to which d-limonene has been added is used for extraction, and the amount of d-limonene added to the ethanol is 5% by volume or more. The anti-influenza effect is characterized by being an influenza-treating effect, as described in [1]. High acetylshikonin content Extract from plants of the Lithospermum genus. [4] It is characterized by containing acetylshikonin. [1] A plant extract of the Lithospermum genus that is high in acetylshikonin, as described above. [ 5 The raw material is characterized by being one of the following Lithospermum species: Lithospermum erythrorhizon Siebold et Zucc., Lithospermum officinale L., Lithospermum arvense L., or Lithospermum zollingeri A.DC. 〔1〕、〔2〕、〔3〕、〔4〕as described in any of High acetylshikonin content a plant extract of the genus Viola.

[0012] 6 A composition for imparting an anti-influenza effect, characterized by containing as a main component the plant extract of the genus Viola described in any of [1], [2], [3], [4], [5]. High acetylshikonin content 7 A composition for imparting an anti-influenza effect, characterized by being labeled with the indication "having an anti-influenza effect", and being any of the compositions for imparting an anti-influenza effect described in 6 8 〔6〕、〔7〕 Food and drink products using the genus Viola, using the composition for imparting an anti-influenza effect described in any of 9 〔6〕、〔7〕 Clothing products using the genus Viola, using the composition for imparting an anti-influenza effect described in any of

[0013] * 10 〔6〕、〔7〕 Miscellaneous goods using the genus Viola, using the composition for imparting an anti-influenza effect described in any of 11 〔6〕、〔7〕 Products using the genus Viola (excluding food and drink products, clothing products, and miscellaneous goods), using the composition for imparting an anti-influenza effect described in any of [Effect of the Invention]

[0014] The High-acetylshikonin-containing Lithospermum plant extract, composition for imparting anti-influenza activity, food and beverages utilizing Lithospermum plants. ​​​​​​​​​​​As described above, these methods allow for the extraction of acetylshikonin and shikonin, the active ingredients of Lithospermum erythrorhizon, at high concentrations. In particular, the extraction method using d-limonene-added ethanol significantly increases the extraction efficiency, enabling highly practical high-concentration extraction. Furthermore, the extracts according to the present invention have been shown to inactivate influenza viruses. This promotes the application and development of various products for influenza prevention and treatment, thereby advancing the effective utilization of Lithospermum erythrorhizon. [Brief explanation of the drawing]

[0015] [Figure 1] This graph shows the component profile of a 70% ethanol extract of Lithospermum erythrorhizon root (A). [Figure 2] This graph shows the component profile of Lithospermum erythrorhizon root ethanol extract (B). [Figure 3] This graph shows the component profile of (C), an ethanol extract containing 5% d-limonene from Lithospermum erythrorhizon root. [Figure 4] This is a photograph of a culture medium showing the anti-influenza virus effect at different extract concentrations of various purple gromwell extracts. [Figure 5] This graph shows the effect of different extract concentrations on the influenza virus. [Modes for carrying out the invention]

[0016] The configuration of the present invention will be outlined below before describing the examples. The basis of this invention is a plant extract of the genus Lithospermum for conferring anti-influenza activity, which includes both influenza prevention and influenza treatment. Ethanol is used for the extraction of this Lithospermum plant extract, but more preferably ethanol to which d-limonene has been added can be used.

[0017] As the raw material for the Lithospermum plant extract of the present invention, any of the following Lithospermum species can be suitably used: Lithospermum erythrorhizon Siebold et Zucc., Lithospermum officinale L., Lithospermum arvense L., or Lithospermum zollingeri A.DC.

[0018] The present invention also includes acetylshikonin derived from Lithospermum plants for imparting anti-influenza activity. In this case, the anti-influenza activity can include both influenza prevention and influenza treatment. Furthermore, the use of ethanol for extraction, more preferably ethanol to which d-limonene has been added, is the same as in the above invention of Lithospermum plant extracts for imparting anti-influenza activity. The same applies to the raw materials as well.

[0019] Furthermore, compositions for imparting anti-influenza activity, primarily composed of the above-mentioned Lithospermum plant extract or acetylshikonin derived from Lithospermum plants, are also within the scope of the present invention. Here, the composition includes all forms of matter, such as liquids, solids, and mixtures of both, and components other than these main components are not particularly limited and can be broadly used. In addition, these compositions for imparting anti-influenza activity may be labeled as having "anti-influenza activity."

[0020] Products using such compositions for imparting anti-influenza activity are also within the scope of the present invention, and include, but are not limited to, foods and beverages using Lithospermum plants, clothing using Lithospermum plants, general merchandise using Lithospermum plants, and pharmaceuticals using Lithospermum plants. Furthermore, these products that bear a label indicating that they "have anti-influenza activity" are also within the scope of the present invention.

[0021] Food and beverages include functional foods and supplements. Clothing includes not only clothing but also personal items such as handkerchiefs, towels, hand towels, masks, and gloves. Miscellaneous goods include, but are not limited to, smartphone and mobile phone covers and straps, paper napkins and paper towels, stationery, toys, disposable tableware, bedding, bags and other bags, and various other items.

[0022] Furthermore, the influenza prevention method and influenza treatment method using the Lithospermum plant extract or acetylshikonin derived from Lithospermum plants described above are also within the scope of the present invention. Moreover, regardless of what the raw material is, acetylshikonin itself for imparting anti-influenza activity is also an invention for which patent rights are claimed. [Examples]

[0023] The following describes examples of the present invention, but the present invention is not limited thereto. Instead of examples, an overview of the research process for developing a novel extraction method for Lithospermum plants and confirming the effects of the extract will be provided.

[0024] [1. Research Topic] Novel extraction method for purple extract with anti-influenza virus activity [2. Summary] We developed a novel extraction method to extract acetylshikonin and shikonin, the active ingredients of Lithospermum erythrorhizon Siebold et Zucc., at high concentrations. We then compared the extraction efficiency of this method with that of existing ethanol extracts to investigate the anti-influenza virus activity of each extract. As a result, we observed inactivation of the influenza virus with the Lithospermum erythrorhizon ethanol extract, and confirmed that this activity was further enhanced by the d-limonene-added ethanol extract, which efficiently extracts acetylshikonin at high concentrations.

[0025] [3. Background] Lithospermum erythrorhizon Siebold et Zucc., a member of the Boraginaceae family, is widely distributed in Japan from Hokkaido to Kyushu, as well as in the mountainous and grassland regions of the Korean Peninsula, China, and the Amur region. Its root (purple root) has been used as a dye and as a medicine exhibiting anti-inflammatory and antitumor effects (Reference 1). Shikonin, a component of purple root, has antiviral effects against influenza A virus (Reference 2), hepatitis C virus (Reference 3), adenovirus (Reference 4), enterovirus (Reference 5), and HIV (Reference 6), and acetylshikonin, a derivative of shikonin, also has anticoxsackievirus effects (Reference 7), so its effective utilization is promising.

[0026] However, due to the lack of established cultivation techniques and the fact that it thrives in cool climates, its population has decreased in response to environmental changes caused by global warming, and as of March 2022, it is designated as Endangered 1B on the Ministry of the Environment's Red List (Ministry of the Environment Red List 2020). Therefore, with the aim of exploring the possibility of further expanding the effective use of Lithospermum erythrorhizon, the Kami Town Medicinal Plant Research Association (established in November 2015) in Kami Town, Kami District, Miyagi Prefecture, has developed a new extraction method for acetylshikonin and shikonin, the active ingredients of Lithospermum erythrorhizon that it cultivates by improving cultivation techniques, with the aim of contributing to their effective use.

[0027] Examples of Lithospermum species used in this invention include Lithospermum erythrorhizon Siebold et Zucc., Lithospermum officinale L., Lithospermum arvense L., and Lithospermum zollingeri A.DC., with Lithospermum erythrorhizon Siebold et Zucc. being preferred. Furthermore, the origin of these Lithospermum species is not particularly limited.

[0028] [4. Experimental Method] [4.-1 Purple sample] The dried roots of Lithospermum erythrorhizon (purple gromwell), cultivated by the Medicinal Plant Research Association of Kami-cho, Kami-gun, Miyagi Prefecture, were used. The moisture content was less than 10%.

[0029] [4.-2 Extraction method] Finely chop the roots of the purple gromwell, and for 500g of that... <1> 70% ethanol 1500mL, <2> Ethanol (95%) 1500mL, <3> After cold immersion for 48 hours in a mixed solution of 1425 mL of ethanol (95%) and 75 mL of d-limonene (d-limonene content: 5 v / v%), the following were filtered and used to obtain purple extract A. <1> ), B( <2> ), C( <3> ) was used. Note that the extraction solution with 70% ethanol is the standard extraction method when using purple as a dye. Also, all reagents used in the test were purchased from Fujifilm Wako Pure Chemical Industries, Ltd. Next, d-limonene was extracted using the same method. Depending on the amount added To investigate whether there were any changes in the composition of the extracts, ethanol (95) extracts were prepared with d-limonene concentrations of 1% (D), 5% (E), 10% (F), and 100% (G), and the concentration of acetylshikonin, the main component, was measured using the method described below.

[0030] [4.-3 Measurement of acetylshikonin and shikonin concentrations in the extract] Three types of purple extracts, A, B, and C, were diluted 10-fold with HPLC-grade ethanol (99.5%) to prepare samples for HPLC measurement. The HPLC measurement conditions were as follows. HPLC measurement conditions HPLC system: SHIMADZU S-20A Mobile phase: 60% Acetonitril, 1% TFA Flow: 1.0 mL / min. Pressure during liquid delivery: 9.1 MPa / cm² 2 inj.Vol.:10uL Column COSMOSIL: 5PE-MS, 4.6 x 250 mm (40°C) Measurement wavelength: 214nm Data processing machine: Chromato PRO RT(min): Shikonin; 5.22~5.33, Acetylshikonin; 7.12~7.32

[0031] [4.-4 Anti-influenza virus activity of Lithospermum extract] [4.-4-1 cells] MDCK (Madin-Darby canine kidney) cell lines (provided by the Sendai Virus Center, Clinical Research Department, Sendai Medical Center) were used. Cells were cultured in DMEM (Dulbecco's Modified Eagle Medium, D5796, Sigma-Aldrich, Inc., St. Louis, MO) supplemented with 10% fetal bovine serum (Thermo Fisher Scientific, Waltham, MA) and penicillin / streptomycin (Fujifilm Wako Pure Chemical Corp., Osaka, Japan).

[0032] [4.-4-2 Virus] Influenza A virus (A / Aichi / 2 / 68, Aichi strain, H3N2 (A Hong Kong) type, provided by the Department of Microbiology, Graduate School of Medicine, Tohoku University) was used. MDCK cells cultured confluently in a T75 flask (BM Bio, Tokyo, Japan) were supplemented with the virus, which was stored at -80°C. Four days after infection, when cytopathic effect (CPE) was observed in many cells, the cells were lysed by freeze-thaw cycles, and the titer of the supernatant after centrifugation (2,000 rpm for 10 minutes) was measured and stored as virus solution at -80°C.

[0033] [4.-4-3 Examination of anti-influenza virus effects] The anti-influenza virus effect was investigated by measuring the plaque-inhibiting effect of the test specimens (References 8-11). Test specimens of purple gromwell extract B and C (hereinafter also referred to as "extract B" and "extract C") were serially diluted 2-fold from 20-fold to 40,960-fold, and an equal volume of virus solution (3 x 10⁻¹) was used. 3After mixing with pfu / mL, the mixture was allowed to react at room temperature for 1 hour. The sample-virus mixture was serially diluted 10-fold from 10-fold to 1,000-fold, and used to infect MDCK cells cultured in monolayers in a 24-well plate (BM Bio) at 37°C under 5% CO2 conditions.

[0034] After 1 hour, the sample-virus mixture was removed, and agar MEM (Nissui Pharmaceutical Co., Ltd., Tokyo, Japan) medium containing 1% Ultrapure agarose (Thermo Fisher Scientific), 5% BSA (Sigma-Aldrich), glutamine (Fujifilm Wako Pure Chemical Corp.), penicillin / streptomycin, 10 mM Hepes (Dojindo, Kumamoto, Japan), 0.01% DEAE-dextran (Cytiva, Marlborough, MA), and 5 μg / mL trypsin (Sigma-Aldrich) was overlaid. The cells were then incubated at 37°C under 5% CO2 for 1.5 days. After fixation with formalin, the agar MEM medium was removed, and the cells were stained with Crystal Violet and the plaques were counted. Using wells without the test sample as a control, the IC50 was calculated from the relationship between the plaque reduction rate and the sample content rate.

[0035] [5. Results] [5.-1 Extraction Method and Acetylshikonin Concentration] Figure 1 is a graph showing the component profile of a 70% ethanol extract of Lithospermum erythrorhizon (A), Figure 2 is a graph showing the component profile of a ethanol extract of Lithospermum erythrorhizon (B), and Figure 3 is a graph showing the component profile of a 5% d-limonene-containing ethanol extract of Lithospermum erythrorhizon (C). Table 1 shows the concentrations of acetylshikonin and shikonin in each Lithospermum extract, and Table 2 shows the change in acetylshikonin concentration in the Lithospermum erythrorhizon ethanol extract with the amount of d-limonene added.

[0036] [Table 1]

[0037] [Table 2]

[0038] As shown in Table 1, the extraction method using d-limonene-added ethanol yielded acetylshikonin at a concentration approximately 230 times higher than that obtained from the 70% ethanol extract, which is the concentration previously used for staining. Furthermore, it yielded approximately four times the concentration compared to the ethanol (95%) extract. In other words, it became clear that the extraction efficiency of acetylshikonin improves with increasing ethanol concentration, and can be further enhanced by adding d-limonene.

[0039] Furthermore, as shown in Table 2, when the amount of d-limonene added to ethanol was increased from 1% to 100%, the amount of acetylshikonin obtained increased to more than three times that of 1% at 5%, more than four times that of 1% at 10%, and more than 40 times that of 1% and more than 14 times that of 5% at 100%. Therefore, it was also revealed that the extraction efficiency of acetylshikonin increases depending on the concentration of d-limonene added.

[0040] [5.-2 Anti-influenza activity] [5.-2-1 Inhibition of plaque formation by novel purple extract] Figure 4 is a photograph of a culture medium showing the anti-influenza virus effect at different extract concentrations of each purple gromwell extract. Figure 5 is a graph showing the influenza virus effect at different extract concentrations of each purple gromwell extract. In Figure 4, the upper row shows extract B and the lower row shows extract C, illustrating plaque formation at extract concentrations of 0.08%, 0.16%, and 0.31%, respectively. As shown, higher extract concentrations (extract concentrations) suppress plaque formation and demonstrate a higher anti-influenza effect.

[0041] [5.-2-2 IC50 values ​​for novel purple extracts] The IC50 values ​​for extracts B and C were 0.086% and 0.007%, respectively. Extract B inactivated over 99.9% of viruses at a 160-fold dilution, 98.0% at a 320-fold dilution, and 87.5% at a 640-fold dilution. Extract C inactivated over 99.9% of viruses at a 160-fold dilution, 99.8% at a 320-fold dilution, 99.2% at a 640-fold dilution, and 97.6% at a 1,280-fold dilution.

[0042] [5.-2-3 IC50 values ​​based on acetylshikonin content] The acetylshikonin content of extracts B and C was 0.85 mg / mL and 4.64 mg / mL, respectively. The IC50 values ​​of extracts B and C, calculated based on acetylshikonin content, were 0.73 μg / mL and 0.31 μg / mL, respectively.

[0043] These results demonstrated that the purple extracts (extracts B and C) possess anti-influenza virus effects. When both extracts were diluted equally, extract C inactivated the virus 12.3 times (0.086 / 0.007) more effectively. However, when diluted based on the amount of acetylshikonin, the difference decreased to 2.4 times (0.73 / 0.31), suggesting that the amount of acetylshikonin in the extract is the main component responsible for virus inactivation.

[0044] As described above, extraction with 5% limonene-containing ethanol (95) was found to be possible at a concentration approximately 230 times higher than that of a 70% ethanol extract, which is the concentration used in conventional staining, and approximately 4 times higher than that of an extract with ethanol (95). Furthermore, the 5% limonene-containing ethanol (95) extract is expected to have applications in various products that have inhibitory effects against influenza viruses.

[0045] <References> 1. Terada, Akira; Tagami, Yasuhiro; Taniguchi, Hiroshige. Chemistry of ancient pigments, shikonin and its derivatives. Organic Synthesis Chemistry 1990; 48:866-875. 2. Zhang Y, Han H, Qiu Het al. Antiviral activity of a synthesized shikonin ester against influenza A (H1N1) virus and insights into its mechanism. Biomed Pharmacother 2017; 93:636-645. 3. Li HM, Tang YL, Zhang ZHet al. Compounds from Arnebia euchroma and their related anti-HCV and antibacterial activities. Planta Med 2012; 78:39-45. 4. Gao H, Liu L, Qu ZYet al. Anti-adenovirus activities of shikonin, a component of Chinese herbal medicine in vitro. Biological & pharmaceutical bulletin 2011; 34:197-202. 5. Zhang Y, Han H, Sun Let al. Antiviral activity of shikonin ester derivative PMM-034 against enterovirus 71 in vitro. Braz J Med Biol Res 2017; 50:e6586. 6. Chen X, Yang L, Zhang Net al. Shikonin, a component of chinese herbal medicine, inhibits chemokine receptor function and suppresses human immunodeficiency virus type 1. Antimicrobial agents and chemotherapy 2003; 47:2810-2816. 7. Liu X, Zhang X, Li Jet al. Effects of Acetylshikonin on the Infection and Replication of Coxsackievirus A16 in Vitro and in Vivo. J Nat Prod 2019; 82:1089-1097. 8. Ikuta K, Mizuta K, Suzutani T. Anti-influenza virus activity of two extracts of the blackcurrant (Ribes nigrum L.) from New Zealand and Poland. Fukushima J Med Sci 2013; 59:35-38. 9. Ikuta K, Hashimoto K, Kaneko H, Mori S, Ohashi K, Suzutani T. Anti-viral and anti-bacterial activities of an extract of blackcurrants (Ribes nigrum L.). Microbiol Immunol 2012; 56:805-809. 10. Sekizawa H, Ikuta K, Ohnishi-Kameyama M, Nishiyama K, Suzutani T. Identification of the Components in a Vaccinium oldhamii Extract Showing Inhibitory Activity against Influenza Virus Adsorption. Foods 2019; 8. 11. Sekizawa H, Ikuta K, Mizuta K, Takechi S, Suzutani T. Relationship between polyphenol content and anti-influenza viral effects of berries. Journal of the science of food and agriculture 2013; 93:2239-2241. [Industrial applicability]

[0046] This invention High-acetylshikonin-containing Lithospermum plant extract, composition for imparting anti-influenza activity, food and beverages utilizing Lithospermum plants. According to the present invention, acetylshikonin and shikonin, which are active ingredients of Lithospermum erythrorhizon, can be extracted at high concentrations, and the extracts according to the present invention have an influenza virus inactivating effect. Therefore, this invention has high industrial applicability in the fields of health, medicine, agriculture, agricultural processing, food, and other industrial fields, as well as all related fields.

Claims

1. A plant extract of the Lithospermum genus that contains a high amount of acetylshikonin for imparting anti-influenza activity, characterized in that ethanol (95) to which d-limonene has been added is used for extraction, and the amount of d-limonene added to the ethanol is 5% by volume or more.

2. The acetylshikonin-rich Lithospermum plant extract according to Claim 1, characterized in that ethanol (95) to which d-limonene has been added is used for extraction, the amount of d-limonene added to the ethanol is 5% by volume or more, and the anti-influenza effect is an influenza preventive effect.

3. The acetylshikonin-rich Lithospermum plant extract according to Claim 1, characterized in that ethanol (95) to which d-limonene has been added is used for extraction, the amount of d-limonene added to the ethanol is 5% by volume or more, and the anti-influenza effect is an influenza-treating effect.

4. The acetylshikonin-rich Lithospermum plant extract according to Claim 1, characterized in that it contains acetylshikonin.

5. The acetylshikonin-rich Lithospermum plant extract according to any one of claims 1, 2, 3, or 4, characterized in that the raw material Lithospermum plant is one of Lithospermum erythrhorhizon Siebold et Zucc., Lithospermum officinale L., Lithospermum arvense L., or Lithospermum zollingeri A.DC.

6. A composition for imparting anti-influenza activity, characterized in that it mainly comprises an acetylshikonin-rich Lithospermum plant extract as described in any one of claims 1, 2, 3, 4, or 5.

7. The composition for imparting anti-influenza activity according to claim 6, characterized in that it is labeled as having "anti-influenza activity."

8. Food and beverages utilizing plants of the genus Lithospermum, using the composition for imparting anti-influenza activity according to any one of claims 6 or 7.

9. Clothing made from plants of the genus Lithospermum, using the composition for imparting anti-influenza activity according to any one of claims 6 or 7.

10. A general merchandise product utilizing a plant of the genus Lithospermum, using the composition for imparting anti-influenza activity described in any one of claims 6 or 7.

11. Products utilizing plants of the genus Lithospermum (excluding food and beverages, clothing, and general merchandise) using the composition for imparting anti-influenza activity described in any one of claims 6 or 7.

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