A mangrove extract, a preparation method thereof and application of the mangrove extract in preparation of anti-inflammatory drugs

Abstract

The application discloses a long-stalked mangrove extract, a preparation method thereof and application of the long-stalked mangrove extract in preparation of anti-inflammatory drugs. The long-stalked mangrove stems or leaves are immersed with ethanol, the immersion liquid is concentrated, then is dispersed with water, and then is extracted with petroleum ether, ethyl acetate and n-butanol in sequence; the extract is concentrated to obtain a petroleum ether extraction part, an ethyl acetate extraction part, an n-butanol extraction part and a water part which is the rest part. The application firstly carries out research on natural active ingredients of the long-stalked mangrove, adopts petroleum ether, ethyl acetate, n-butanol and water as solvents, carries out distribution extraction on the stems and leaves of the long-stalked mangrove, and then evaluates anti-tumor, anti-inflammatory and anti-oxidation activities of each extraction part, so as to find effective parts and pharmacological activities of the long-stalked mangrove. The research result can not only provide a reference for extraction of natural plant effective ingredients by different extraction agents, but also provide important guiding significance for comprehensive development of the long-stalked mangrove resources.

Description

Technical Field

[0001] This invention belongs to the field of biomedicine, specifically relating to an extract of *Magnolia denudata*, its preparation method, and its application in the preparation of anti-inflammatory drugs. Background technology:

[0002] *Manglietia longipedunculata* Zeng et Law is an evergreen broad-leaved plant belonging to the genus *Manglietia* in the family Magnoliaceae. It typically grows in forests at altitudes of 650-800 meters. With excellent timber quality, it is an outstanding ornamental and timber species with high economic value. Due to a lack of pollinating insects, asynchronous development of stamens and pistils, and an extremely short receptive period of the stigma, reproduction is very difficult; in its natural state, it essentially only flowers and does not bear fruit. Only one population of *Manglietia longipedunculata* has been found worldwide in the Nankun Mountain Provincial Nature Reserve. This population is extremely small and of very limited size, classifying it as a rare and endangered plant.

[0003] Currently, research on Magnolia longipes mainly focuses on its artificial propagation and introduction cultivation, while research on the efficacy and utilization of Magnolia longipes has not yet been reported. Summary of the Invention:

[0004] The purpose of this invention is to provide an extract of *Magnolia denudata*, its preparation method, and its application in the preparation of anti-inflammatory drugs.

[0005] The extract of Magnolia longipes of the present invention is obtained by extracting the stems or leaves of Magnolia longipes with ethanol, dispersing the extract with water after concentration, and then extracting with petroleum ether and ethyl acetate in sequence. The extracts are then concentrated to obtain the petroleum ether fraction and the ethyl acetate fraction, respectively.

[0006] Preferably, the concentration is vacuum concentration under reduced pressure, and the temperature does not exceed 60°C.

[0007] The present invention also provides the application of the above-mentioned *Magnolia denudata* extract in the preparation of anti-inflammatory drugs.

[0008] The present invention also provides an anti-inflammatory drug comprising the aforementioned *Magnolia denudata* extract as an active ingredient.

[0009] Preferably, the part is the petroleum ether part or ethyl acetate part of the stem or leaves of Magnolia longipes.

[0010] This invention is the first to study the natural active ingredients of *Magnolia longipes*. Petroleum ether, ethyl acetate, n-butanol, and water were used as solvents for the distribution extraction of the stems and leaves of *Magnolia longipes*. The anti-inflammatory activity of each extracted fraction was then evaluated to identify the effective components and pharmacological activities of *Magnolia longipes*. The research results not only provide a reference for the extraction of effective components from natural plants using different extractants but also offer important guidance for the comprehensive development of *Magnolia longipes* resources. Attached Figure Description

[0011] Figure 1 It is the anti-inflammatory activity of different extracts from Magnolia longipes. Detailed implementation method:

[0012] The following embodiments are further illustrations of the present invention, but not limitations thereof.

[0013] Example 1:

[0014] Experimental Procedures and Results

[0015] 1. Preparation of crude extracts from stems and leaves of *Magnolia longipes* using different solvents

[0016] 1.1 Preparation of fresh green leaves of Magnolia longipes

[0017] First, 100g of fresh green leaves from *Magnolia longipes* were soaked in 300mL of anhydrous ethanol for 24 hours, followed by concentration of the extract. This soaking process was repeated three times. The three ethanol extracts were then combined and concentrated under vacuum (temperature not exceeding 60℃). The concentrated extract was dispersed in water, first extracted three times with petroleum ether to obtain the petroleum ether extract, then three times with ethyl acetate to obtain the ethyl acetate extract, and finally extracted with n-butanol to obtain the n-butanol extract. The remaining fraction was the aqueous fraction. Each fraction was then concentrated under vacuum (temperature not exceeding 60℃).

[0018] 1.2 Preparation of crude extracts from fresh stems and branches of Magnolia longipes

[0019] First, 100g of fresh stems and branches of *Magnolia longipes* were soaked in 300mL of anhydrous ethanol for 24 hours, followed by concentration of the extract. This soaking process was repeated three times. The three ethanol extracts were then combined and concentrated under vacuum (temperature not exceeding 60℃). The concentrated extract was dispersed in water, first extracted three times with petroleum ether to obtain the petroleum ether extract, then three times with ethyl acetate to obtain the ethyl acetate extract, and finally extracted with n-butanol to obtain the n-butanol extract. The remaining fraction was the aqueous fraction. Each fraction was then concentrated under vacuum (temperature not exceeding 60℃).

[0020] 1.3 Anti-inflammatory activity test

[0021] RAW264.7 cells were grown at a rate of 9 × 10⁻⁶.5 Cells were seeded at a density of 10 cells / well in 96-well plates. After 24 hours of adhesion, samples were added, followed by 2 hours of treatment. Then, 10 μL of LPS (100 μg / mL working solution) was added to each well, and the plates were incubated at 37°C for 24 hours. 90 μL of cell supernatant was then transferred to another 96-well plate. 45 μL of Griess I reagent and 45 μL of Griess II reagent were added to each well, and the OD value was measured at 540 nm. The inhibition rate was calculated based on the OD value.

[0022] The anti-inflammatory activity test results of extracts of Magnolia longipes in different solvents are shown in Table 1 and Figure 1 Table 1 shows that all eight different extracts from the stems and leaves exhibited varying degrees of anti-inflammatory activity. Among the four stem extracts, the ethyl acetate extract showed the strongest anti-inflammatory activity, inhibiting NO formation by 92.99% at a concentration of 1 mg / mL; followed by the petroleum ether fraction with an inhibition rate of 87.15%; the n-butanol fraction with an inhibition rate of 84.50%; and the aqueous fraction showed weak anti-inflammatory activity with an inhibition rate of 53.30%. Similarly, among the four leaf extracts, the ethyl acetate extract showed the strongest anti-inflammatory activity, inhibiting NO formation by 93.81% at a concentration of 1 mg / mL; followed by the petroleum ether fraction with an inhibition rate of 71.93%; the n-butanol fraction with an inhibition rate of 61.12%; and the aqueous fraction showed weak anti-inflammatory activity with an inhibition rate of 39.80%. Experimental results showed that the petroleum ether, ethyl acetate, and n-butanol fractions of the stems and leaves of *Magnolia longipes* all exhibited good anti-inflammatory activity, even superior to the positive control indomethacin. In particular, the ethyl acetate fraction of the stems and leaves of *Magnolia longipes* showed strong anti-inflammatory activity, providing new plant resources for the discovery of lead compounds for anti-inflammatory drugs.

[0023] Table 1. Anti-inflammatory activity (inhibition rate %) of different solvent extracts from the stems and leaves of *Magnolia longipes*.

[0024]

[0025] Further investigation of the anti-inflammatory activity of Magnolia longipes IC 50 The values ​​were determined, with indomethacin as the positive control. The results are shown in Table 2. The petroleum ether and ethyl acetate fractions of *Magnolia longipes* stems showed strong anti-inflammatory activity, with an IC50 value of [value missing]. 50 The values ​​were 7.20±0.22 and 9.01±0.33 μg / mL, respectively, which were superior to the positive control indomethacin; the n-butanol extract from the stem showed weak anti-inflammatory activity, while its aqueous portion showed no anti-inflammatory activity. Furthermore, among the four extracts from the leaves of *Magnolia longipes*, the petroleum ether and ethyl acetate extracts showed better anti-inflammatory activity, with their IC50 values ​​being 7.20±0.22 and 9.01±0.33 μg / mL, respectively. 50The values ​​were 14.96±0.61 and 15.34±1.78 μg / mL, respectively, while n-butanol and water showed weak or no anti-inflammatory activity. This further indicates that the anti-inflammatory active substances of *Magnolia longipes* are mainly concentrated in the small polar parts of the stem and leaves.

[0026] Table 2. Anti-inflammatory activity of different solvent extracts from the stems and leaves of *Magnolia denudata* (IC50). 50 (μg / mL)

[0027]

[0028]

Claims

1. A method for preparing an anti-inflammatory extract of Magnolia longipes, characterized in that, The extract is obtained by ethanol extraction of the stems or leaves of Magnolia longipes, followed by concentration and dispersion in water, and then extraction with petroleum ether and ethyl acetate in sequence. The extracts are then concentrated to obtain the petroleum ether fraction and the ethyl acetate fraction, respectively. The Magnolia longipes extract is the petroleum ether fraction or the ethyl acetate fraction of the stems or leaves of Magnolia longipes.

2. The preparation method according to claim 1, characterized in that, The concentration described is vacuum concentration under reduced pressure, with the temperature not exceeding 60°C. o C.

3. An extract of *Magnolia denudata* prepared according to the preparation method described in claim 1 or 2.

4. The use of the long-stemmed magnolia extract according to claim 3 in the preparation of anti-inflammatory drugs.

5. An anti-inflammatory drug, characterized in that, The extract of *Magnolia denudata* as described in claim 3 is used as the active ingredient.

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