阅读说明：本技术 桃金娘果中间苯三酚衍生物的快速制备及应用 (Rapid preparation and application of phloroglucinol derivatives in myrtle fruits ) 是由 徐明行 万思梦 姚丽媛 冷迎慧 罗碧 袁云飞 于 2020-09-12 设计创作，主要内容包括：本发明涉及天然有机化学领域,提供了间苯三酚衍生物的快速制备及应用,特别涉及桃金娘果中间苯三酚衍生物的快速制备及应用。其特征在于：桃金娘果粉碎物乙醇提取、萃取后得到的正己烷部浸膏、乙酸乙酯部浸膏、正丁醇部浸膏、水部浸膏,通过一些手段检测确定间苯三酚富集的部位为正己烷部,正己烷部通过硅胶柱梯度洗脱,得到的组分经过检测确定间苯三酚富集的部位再进行分离,通过可以快速分离到间苯三酚衍生物,仅从一个间苯三酚富集的小组分中就分离到了7个间苯三酚衍生物。该发明首次发现了桃金娘果提取物含有间苯三酚衍生物且具有抗S.aureus(革兰氏阳性菌)的活性,为制备治疗MASA提供了新的安全植物和天然化合物,为制备抗MASA的药物或药物组合物提供有力的技术支持。该发明提供了快速制备、分离间苯三酚衍生物的方法,大大提高了分离的效率。(The invention relates to the field of natural organic chemistry, provides rapid preparation and application of phloroglucinol derivatives, and particularly relates to rapid preparation and application of phloroglucinol derivatives in myrtle fruits. The method is characterized in that: the method comprises the steps of extracting crushed myrtle fruits by ethanol to obtain n-hexane part extract, ethyl acetate part extract, n-butanol part extract and water part extract, detecting and determining that a phloroglucinol-enriched part is an n-hexane part by some means, carrying out gradient elution on the n-hexane part by a silica gel column, detecting and determining the phloroglucinol-enriched part of the obtained components, then separating, and quickly separating phloroglucinol derivatives to obtain 7 phloroglucinol derivatives from only one phloroglucinol-enriched small component. The invention discovers for the first time that the myrtle fruit extract contains phloroglucinol derivatives and has the activity of resisting S.aureus (gram positive bacteria), provides new safe plants and natural compounds for preparing MASA and provides powerful technical support for preparing MASA-resisting medicines or medicinal compositions. The invention provides a method for rapidly preparing and separating phloroglucinol derivatives, and greatly improves the separation efficiency.)

1. The rapid preparation and application of phloroglucinol derivatives in myrtle fruits are characterized in that: the phloroglucinol derivative in the myrtle fruits can be rapidly prepared, and the application thereof is provided.

2. Phloroglucinol derivatives according to claim 1, characterized in that: has a basic skeleton structure shown in a formula I:

3. the rapid preparation according to claim 1, characterized in that: after extraction, the phloroglucinol derivative is positioned by an activity test method.

4. The activity test method according to claim 3, characterized in that: assaying for activity against s.

5. The rapid preparation according to claim 1, characterized in that: in the separation process, ultraviolet rays are focused, and TLC develops a compound with red color.

6. Use according to claim 1, characterized in that it is mainly used in the antibacterial field.

Technical Field

The invention relates to rapid preparation and application of phloroglucinol derivatives, in particular to rapid preparation and application of phloroglucinol derivatives in myrtle fruits.

Background

There are about 18 kinds of plants of myrtle (Myrtaceae) worldwide, the plants are mainly distributed in tropical regions of Asia and oceanic continents, and only 1 kind of myrtle (Rhodomyrtus tomentosa (Ait) Hassk) is in China. The myrtle enjoys humid and high-temperature climatic environment, is native to the south and southeast asia, and is distributed in india, the southeast to the south of china, philippines, the south to malaysia and the island of sura westward. Myrtle is commonly used as a herbal remedy for treating or preventing diseases in chinese folks, particularly in southern regions. People in Tang Dynasty have found good medicinal value, and the compendium of materia Medica is also included. In 1765, the record in Bencao gang mu Shi Yi (supplement to compendium of materia Medica) of the Ming Dynasty Zhao school: "Guangdong Zhi: the roses named as the mother include myrtle, rosette, plum, slightly sharp, peach-like, purplish middle stem, deep yellow silk like gold millet, named as myrtle, eight and september, cyanosis, milk cow, Guilin, and more of the Jinguangzhou. Yue ge yun: the flower-picking basket is full of flowers, the exotic love is to seek children, and the boy love is the myrtle. Flower mirror: the hypericum is a myrtle which is seen in Guilin county, has a flower like peach and big, is more colorful, has pure purple middle stem and yellow pithy, spreads out flowers, and is just like a gold wire, and is true and ripe in eight and September. Cyanotic bovine meat is milky and sweet in taste, and can be used as a medicine. For example, when the root is split, the root still covered with earth, and the transplanting can be performed in the next year. The flower moves blood. The seed flavor is sweet and enters spleen, and nourishes blood and improves eyesight. "1975 edition (second edition)" national Chinese herbal medicine compilation "records: "root: dispelling pathogenic wind, activating collaterals, astringing, and relieving diarrhea; can be used for treating acute and chronic gastroenteritis, gastralgia, dyspepsia, hepatitis, dysentery, rheumarthritis, psoatic strain, functional uterine bleeding, and rectocele; it is used externally to treat burns and scalds. Leaf: astringe to check diarrhea, stop bleeding. Can be used for treating acute gastroenteritis, dyspepsia, and dysentery, and can be used for treating traumatic hemorrhage by external application. And (4) fruit: tonify blood, nourish yin, prevent abortion. Can be used for treating anemia, asthenia after illness, neurasthenia, tinnitus, and spermatorrhea. "the Song immediately written" Chinese materia Medica "in 1999: to nourish blood and stop bleeding, astringe intestines to secure essence. Treating blood deficiency, asthenia, hematemesis, epistaxis, internal injury, hemoptysis, hematochezia, metrorrhagia, spermatorrhea, leukorrhagia, dysentery, rectocele, scald, and traumatic hemorrhage. There are also medical records related to Vietnam in traditional medicine, such as immature fruits used to treat diarrhea or dysentery, mature fruits used to stimulate the immune system, etc. (Florida search database). Myrtle is widely planted in tropical and subtropical regions, and its fruits are used for brewing wine, and also made into jelly, or syrup-added fresh cans for human consumption in china and vietnam. The fruit can also be used as pie or jam. In recent years, due to the beautiful appearance of the flower color, the plant height is suitable, and the application range is expanding from medicinal plants to ornamental plants. In addition, it has also proven to be a promising flame retardant for use in the fire section of Himalayan mountains.

Modern pharmacological studies show that: the myrtle has various pharmacological activities of resisting bacteria, tumors, malaria and the like. However, it is not included in the pharmacopoeia of the people's republic of China, 2015 edition. The research value and the potential of the traditional Chinese medicine and the western medicine of the myrtle are great, and the use value of the western medicine is yet to be developed. Besides, the myrtle also has wide application value. Further research finds that the active component of the myrtle is phloroglucinol compounds. However, whether the fruits of myrtle contain the compounds or not, how to efficiently separate and prepare the novel phloroglucinol derivatives with corresponding pharmaceutical activities is not reported in the prior art.

Disclosure of Invention

The invention aims to provide rapid preparation and application of phloroglucinol derivatives in myrtle fruits.

The invention aims to provide the rapid preparation and application of phloroglucinol derivatives in myrtle fruits, which are realized by the following technical scheme:

phloroglucinol derivatives, refer to compounds having a skeleton of the general formula i as shown below:

the rapid preparation method of the phloroglucinol derivative comprises the following steps:

the crushed myrtle is soaked in ethanol for four times and concentrated to obtain ethanol extract. Then extracting with n-hexane, ethyl acetate and n-butanol for three times respectively to obtain n-hexane extract, ethyl acetate extract, n-butanol extract and water extract.

Activity detection shows that the petroleum ether extract has good activity against S.aureus (gram-positive bacteria), and that phloroglucinol contained in the petroleum ether extract is more than that contained in the ethyl acetate extract, the n-butanol extract and the water extract, so that the petroleum ether extract is selected for the next experiment.

Further, it is known that most phloroglucinol derivatives are excellent in s.aureus (gram positive bacteria) resistance, and therefore, attention is focused on the s.aureus (gram positive bacteria) activity, which is an index of the phloroglucinol derivative content in a crude extract.

Taking the n-hexane part extract to pass through a normal phase silica gel column n-hexane: ethyl acetate (100: 0-0: 100), ethyl acetate: gradient eluting with methanol (20: 1-0: 100) to obtain 11 components Fr.A-Fr.K.

Further, when combining, focus was put on uv, and uv and TLC developed the same fractions were combined.

Further, the Thin Layer Chromatography (TLC) identification method is: observing under an ultraviolet lamp at 254nm, wherein the developer is concentrated sulfuric acid-vanillin, soaking, heating with a 120 deg.C blower until spots appear, and reducing the spots to red to obtain phloroglucinol derivatives.

The activity of Fr.A-Fr.K11 fractions was measured to show that the anti-S.aureus (gram positive bacteria) activity of Fr.D, Fr.E, Fr.F and Fr.G could reach nm level, so these sites were preferentially done.

And Fr.D, Fr.E, Fr.F and Fr.G, by comprehensively using separation means such as normal phase silica gel chromatography, reverse phase silica gel chromatography, HPLC and the like, and identifying by Thin Layer Chromatography (TLC), separating the monomeric compounds, and analyzing and identifying the structures of the monomeric compounds by mass spectrometry, ultraviolet spectroscopy, infrared spectroscopy, nuclear magnetic resonance and the like.

The following compounds were obtained from the fr.e component only:

the invention relates to a rapid preparation method and application of phloroglucinol derivatives in myrtle fruits. Compared with the prior art, the rapid preparation and application of phloroglucinol derivatives in myrtle fruits have the remarkable advantages that:

the invention discovers for the first time that myrtle fruits and extracts thereof can be used for resisting natural S.aureus (gram positive bacteria) activity, crude extracts of n-hexane parts have the highest activity, and main components of myrtle fruits and extracts thereof for resisting S.aureus (gram positive bacteria) are phloroglucinol derivatives.

The invention provides a method for rapidly positioning a phloroglucinol derivative, wherein the structure of the phloroglucinol derivative is detected roughly by distinguishing TLC ultraviolet absorption condition and concentrated sulfuric acid vanillin color development condition in the prior art, and if the structure contains too many impurities, the phloroglucinol derivative cannot be found by simple TLC; if the crude extract is directly detected by HPLC, the HPLC column is clogged by too many impurities contained in the sample, and the crude plant extract contains more impurities relative to the phloroglucinol derivative, the presence of the phloroglucinol derivative may not be detected by a small amount of the sample, and the crude extract is complicated in composition and difficult to judge whether the extract is a phloroglucinol derivative only by ultraviolet and retention time.

The invention adopts the activity of anti-S.aureus as an index, and the positioning is more visual and clear by distinguishing the TLC ultraviolet absorption condition and the concentrated sulfuric acid vanillin coloration condition, thereby greatly accelerating the extraction and separation speed in the extraction process.

The invention can be applied to the scientific research work of the phloroglucinol derivatives of other plants, and can accelerate the scientific research of the phloroglucinol derivatives; the rapid preparation method can be applied to industrial production, is beneficial to enterprises to obtain phloroglucinol derivative antibacterial extracts with higher purity and better activity, and greatly accelerates the progress of industrial production; in view of the wide biological activity of the phloroglucinol derivatives, the invention can be widely applied to various preparation fields, such as antibiosis, tumor resistance, antivirus, anti-inflammation, antimalarial, gastric ulcer resistance, antioxidation, antirheumatic, uvioresistant, skin care, psoriasis, blood sugar reduction, neuritis resistance, bone cell differentiation promotion and the like.

Drawings

FIG. 1 is a structural formula of Rhodomyrtone;

FIG. 2 is Rhodomyrtone¹A graph of H-NMR;

FIG. 3 is Rhodomyrtone¹A graph of C-NMR;

FIG. 4 is a structural formula of Tomentodenion C;

FIG. 5 is a view of Tomentodeion C¹A graph of H-NMR;

FIG. 6 is a view of Tomentodeion C¹A graph of C-NMR;

FIG. 7 is a structural formula of Tomentodene B;

FIG. 8 is a view of Tomentodione B¹A graph of H-NMR;

FIG. 9 is a view of Tomentodione B¹A graph of C-NMR;

FIG. 10 is the structural formula of Calliviminone E;

FIG. 11 is of Calliviminone E¹A graph of H-NMR;

FIG. 12 is of Calliviminone E¹C-NMR chart;

FIG. 13 is a structural formula of 8a-hydroxy-3,3,6,6,8,8-hexamethyl-8,8 a-dihydrobenzoc [ c ] [1,2] dioxine-5,7(3H,6H) -dione;

FIG. 14 shows 8a-hydroxy-3,3,6,6,8,8-hexamethyl-8,8 a-dihydrobenzol [ c ]][1,2]Process for preparing dioxine-5,7(3H,6H) -dion¹A graph of H-NMR;

FIG. 15 shows 8a-hydroxy-3,3,6,6,8,8-hexamethyl-8,8 a-dihydrobenzol [ c ]][1,2]Process for preparing dioxine-5,7(3H,6H) -dion¹A graph of C-NMR;

FIG. 16 is the structural formula of Callistenone B;

FIG. 17 is of Callistenone B¹A graph of H-NMR;

FIG. 18 is of Callistenone B¹A graph of C-NMR;

FIG. 19 is the structural formula of Isopyrtucommulone B;

FIG. 20 shows Isogyrucommalone B¹A graph of H-NMR;

FIG. 21 shows Isomethyltucommulone B¹A graph of C-NMR;

Detailed Description

The following will describe in detail the rapid preparation and application of phloroglucinol derivatives from myrtle fruits according to the present invention, with reference to preferred embodiments.

Example 1 about 100kg of wet myrtle fruits harvested in Ganzhou, Jiangxi province of China are dried by a small-sized dryer and then repeatedly aired in the sun for three days to obtain 35kg of dried myrtle fruits, the dried myrtle fruits are crushed by a small-sized crusher to obtain coarse powder, the coarse powder is extracted by 95% ethanol (75L) for four times, the first three times are 12 hours and the last time is 24 hours, and the total extract obtained by spin-drying the extracting solution by a rotary evaporator is about 3 kg. Then extracting with petroleum ether, ethyl acetate and n-butanol to obtain petroleum ether extract (441.6g), ethyl acetate extract, n-butanol extract and water extract.

Test example: the total crude extract of Myrtus communis, petroleum ether part, ethyl acetate part, n-butanol part, and water part have in vitro antibacterial activity.

Positive control drug: kanamycin (Kanamycin), Vancomycin (Vancomycin).

Negative control drug: blank, DMSO

Indicator (b): resazurin (Sigma).

Test strains: au reus (CMCC 26003), e.coli (ATCC 8739) were purchased from guangdong institute of microorganisms.

Culture medium: MH (hydrolyzed casein peptone) culture medium

Experimental reagent: DMSO, ultrapure water (MILLIPORE, USA), agar.

An experimental instrument: analytical balance (SARTORIUS, germany), clean bench (shanghai zhicheng analytical instruments manufacturing ltd), pressure steam sterilization pot (shanghai sanshen medical instruments ltd), constant temperature incubator (SANYO, japan), constant temperature shaking incubator (shanghai-constant technology ltd), general refrigerator (qingdao hel electric appliances ltd), ultraviolet visible spectrophotometer (shanghai analytical instruments ltd), 96-well cell culture plate (CORNING, usa), and the like.

The experimental method comprises the following steps: the experiment adopts a resazurin color development method to determine the bacteriostatic ability of the compound, and uses the Minimum Inhibitory Concentration (MIC) value to reflect the bacteriostatic effect of the compound. Resazurin is a blue non-fluorescent redox indicator, and various reductases in living cells can change the indicator from blue to pink, but inactive cells cannot reduce due to their non-metabolic capability, and the antibacterial ability of the compound is determined by observing the change in the color of bacterial suspension. After the indicator, the compound and the bacterial liquid are co-cultured for a certain time, if the bacterial liquid turns red, the compound has no bacteriostatic activity, and if the bacterial liquid maintains blue, the compound has bacteriostatic activity.

The experimental steps are as follows: recovering strains: and (3) respectively coating various frozen strains on an MHB solid culture medium, and putting the MHB solid culture medium in a constant temperature incubator at 37 ℃ for overnight culture.

Culturing test bacterial liquid: individual colonies were selected and inoculated into MHB liquid medium, and then cultured in a 37 ℃ incubator with shaking at constant temperature for 12 hours. Diluting the propagated and grown strain with MHB culture medium, and adjusting OD 600 value to about 0.07 with ultraviolet spectrophotometer to obtain 1.25+106 CFU/mL bacterial liquid. Gradient dilution method: firstly, mixing the resazurin indicator with a purified water solution in proportion until the concentration is 100 mu g/mL, mixing the prepared resazurin solution with a bacterial solution to be detected in a ratio of 1.5: 1, the compound is prepared with DMSO at a concentration of 500mg/mL, 180. mu.L of the homogenate and 20. mu.L of the compound are added to the first row, and 100. mu.L of each of the remaining rows are added, followed by dilution in a gradient. And finally, placing the 96-well plate in a constant-temperature incubator at 37 ℃ for culturing for 10-12 hours, and observing color change.

Results of crude extracts against gram-positive and gram-negative bacteria:

and separating the petroleum ether part extract. Subjecting the petroleum ether part extract to normal phase silica gel column n-hexane: ethyl acetate (100: 0-0: 100), ethyl acetate: methanol (20: 1-0: 100) is used for gradient elution to obtain Fr.A-K11 components.

Test example: the in vitro antibacterial activity of the separated petroleum ether components Fr.A, Fr.B, Fr.C, Fr.D, Fr.E, Fr.F, Fr.G, Fr.H, Fr.I, Fr.J and Fr.K of myrtle fruit.

Positive control drug: kanamycin (Kanamycin), Vancomycin (Vancomycin).

The experimental method still adopts a resazurin color development method to determine the bacteriostatic ability of the compound.

Results of Fr.A-K against gram-positive and gram-negative bacteria

And Fr.A is detected by TLC thin-layer chromatography and vanillin sulfate color development to show that most of the fat chains are fat chains. And most of the fatty acid is detected by TLC thin-layer chromatography and vanillin sulfate color development detection of the protein Fr.B and Fr.C.

The following phloroglucinol derivatives were obtained by selecting only one fr.e fraction with moderate activity for separation:

rhodomyrtone, the chemical structure of which is shown in FIG. 1; the molecular formula is as follows: c₂₆H₃₄O₆. As shown in fig. 2, nuclear magnetic 1H NMR (cd3cl3,500MHz): δ H6.15 (1H, s, H-5),4.30(1H, s,9-H),3.01(2H, m, J ═ 3Hz, H-2'),2.30(1H, m, J ═ 2.30Hz, H-3'),1.57 (3H, s,4-CH3),1.46(3H, s,4-CH3),1.43(3H, s,2-CH3),1.40(3H, s,2-CH3),0.90(6H, d, J ═ 1.01Hz, H-3, H-4'), 0.85(6H, dd, J ═ 0.9Hz, H-3 ″, H-4 "); as shown in FIG. 3, 13C-NMR (CD3Cl3,125MHz): delta C212.09 (C-3),206.53 (C-1'),198.11(C-1),167.31(C-4a),162.63(C-8),158.58(C-6),155.66(C-10a),114.27(C-9a),107.69(C-7),106.53 (C-8a),94.85(C-5),56.05(C-2),53.19(C-2'),47.22(C-4),45.83(C-1"),25.24(C-9),25.19(C-2"), 25.1.1 ″, and4(C-4), 24.74(C-4),24.60(C-2),24.17(C-2),23.52(C-2"),23.19(C-3'),22.81(C-3'),22.75(C-2").

tomentodenion C, the chemical structure of which is shown in FIG. 4; the molecular formula is as follows: c₂₆H₃₄O₆. As shown in fig. 5, 1H NMR (cd3cl3,500mhz): δ H1.54 (1H, m, H-1),1.44(2H, m, H-2),2.03(2H, dd, J ═ 15.5,10.0Hz, H-3a),2.11(1H, m, H-5),1.72(2H, m, H-6),2.38(2H, m, H-7),2.59(1H, dd, J ═ 18.0,9.0Hz, H-9),1.73(2H, m, H-10),0.90(3H, s, H-12),0.99(3H, s, H-13),1.26(1H, s, H-14),4.85(2H, br, s, H-15),2.99(3H, dd, J ═ 8, H-8, 1.5H ', 1.8, 1H-8, 1H', 49H ', 8, 1, 1.0.0, 8, 1H-5, H', 49 ',8', 1.67(3H, m,9'),0.97(3H, d, J ═ 6.5Hz,10'),0.90(1H, d, J ═ 6.6Hz,11'),0.90(2H, s,12'),1.38 (1H, s,13'),1.36(3H, s,14'),1.35(3H, s,15'), as shown in fig. 6, 13C-NMR (cd3cl3,125mhz): δ C213.86 (C-3'), 197.90(C-5'),169.40(C-1'),154.56(C-8),114.66(C-6'),109.75(C-15),85.43(C-4),56.94(C-1),55.38(C-4'),47.81 (C-2'),42.75(C-9),41.96(C-8'),41.47(C-3),38.52(C-5), 37.38 (C-36), 10.7 '),36), 33.35(C-11),29.50(C-12), 29.18(C-7'),27.45(C-9'),26.08(C-13'),25.34(C-12'),25.02(C-15'),24.15(C-10'),23.59(C-14),23.28(C-2),22.80 (C-14'),22.13(C-13'),21.81(C-11').

Tomentodione B, the chemical structure of which is shown in FIG. 7; the molecular formula is as follows: c₂₆H₃₄O₆. As shown in fig. 8, 1H NMR (cd3cl3,500mhz): δ H1.50 (1H, m, H-1),1.56(2H, m, H-2),2.02(2H, dd, J ═ 15.5,10.0Hz, H-3a),1.74(1H, m, H-5),1.67(2H, m, H-6),2.34(2H, m, H-7),2.40(1H, dd, J ═ 18.0,9.0Hz, H-9),1.74(2H, m, H-10),0.95(3H, s, H-12),0.98(3H, s, H-13),1.34(1H, s, H-14),4.92(2H, br, s, H-15),2.89(3H, dd, J ═ 0.8, 5H-8, 1.7, 1H-8, 1H ', 8, 1.8, 1H-8, 1, 8'd, 1, 8H-8'd, 1, 8H-7, 1, 8H-9 a, H-9',8 ', 1.48(3H, m,9'),0.84(3H, d, J ═ 6.5Hz,10'),0.98(1H, d, J ═ 6.6Hz,11'),1.33(2H, s,12'),1.36 (1H, s,13'),1.34(3H, s,14'),1.35(3H, s,15 '); as shown in FIG. 9, 13C-NMR (CD3Cl3,125MHz): delta C213.83 (C-3'), 197.15(C-5'),170.28(C-1'),150.79(C-8),116.39(C-6'),111.00(C-15),86.33(C-4),58.09(C-1),55.44(C-4'),47.62 (C-2'),46.21(C-9),42.30(C-8'),40.90(C-3),39.77(C-5),36.14(C-10),35.35(C-7),34.41(C-11),29.74(C-12), 28.35(C-7'),26.59(C-9'),25.44(C-13'),25.38(C-12'),25.27(C-15'),24.89(C-15'),24.63 (C-10).'),24.13(C-14), 23.75(C-2),23.36(C-14'),21.78(C-13'),21.60(C-11').

Calliviminone E, the chemical structure of which is shown in figure 10; the molecular formula is as follows: c₂₅H₃₈O₃. As shown in fig. 11, 1H NMR (cd3cl3,500mhz): δ H2.30 (m, H-7),1.32(m, H-8),0.71(m, H-8),1.58(m, H-9),0.86(d, J ═ 7.0Hz, H-10),0.88(d, J ═ 7.0Hz, H-11), 1.40(s, H-12),1.37(s, H-13),1.40(s, H-14),1.36(s, H-15),2.49(dd, J ═ 17.5,1.5Hz, H-1'),2.16(m, H-1'),5.33(br s, H-2'),2.14(m, H-4'),2.02(m, H-6'),1.94(m, H-6'),2.04(m, H-6, t ═ 7, H-9, H-7, H-13, H-15, H-1, H-15, H-6', 2.06, H-6, H-6, H-1, H-6, H, 1.59 (s, H-10'). As shown in FIG. 12, 13C-NMR (CD3Cl3,125MHz): delta.C 212.9(C-3),208.15(C-1),208.15(C-2),136.37(C-3'), 131.58(C-8'),123.93(C-7'),115.94(C-2'),67.59(C-6),56.51(C-9),56.19(C-4),39.02(C-8),37.14(C-5'),33.90 (C-7),30.54(4'),29.31(C-1'),26.31(C-6'),26.13(C-15),25.93(C-12),25.71(9'),25.40(C-6),24.93(C-14),24.34 (C-10),24.27(C-13),20.90(C-11),17.74(10').

8a-hydroxy-3,3,6,6,8,8-hexamethyl-8,8a-dihydrobenzo[c][1,2]dioxine-5,7(3H,6H) -dione, the chemical structure of which is shown in FIG. 13; molecular formula C₁₄H₂₀O₅. As shown in FIG. 14, 1H NMR (CD3Cl3,500MHz): delta H1.07(3H, s,8a-CH3),1.35(3H, s, 8b-CH3),1.38(3H, s,3a-CH3),1.39(6H, s,6-CH3),1.52((3H, s,3b-CH3),7.17(1H, s, H-4) as shown in FIG. 15, 13C-NMR (CD3Cl3,125MHz): delta C210.65 (C-5,7),198.36(C-5,7),143.05(C-4),131.66(C-4a),97.38(C-8a), 79.09(C-3),54.97(C-8,6),51.65(C-8,6),26.60(-CH3),24.07(-CH3),23.90(-CH3), 59648-CH 3),20.93(-CH3), 20.15 (-CH 2), 59617).

Callistenone B, the chemical structure of which is shown in FIG. 16; the molecular formula is as follows: c₂₅H₃₂O₆. As shown in fig. 17, 1H NMR (cd3cl3,500mhz): δ H6.31 (1H, s, H-7),4.29(1H, d, J ═ 4.3Hz, H-9),1.39(3H, s, H-10),1.33(3H, s, H-11),1.68(3H, s, H-12),1.50 (3H, s, H-13),3.27(1H, dd, J ═ 13.8,6.6Hz, H-2'),3.01(1H, dd, J ═ 13.8,6.6Hz, H-2'),2.30-2.38(1H, m, J ═ 4.3Hz, H-3'),1.02(3H, d, J ═ 6.6Hz, H-4'),0.99(3H, d, J ═ 6.6Hz, J ═ 4, H-3H ', 1.89H, 1.5H-6H, 1.9, 1H-2, 1.6 Hz, 89, 1H-2, 1.9, H-1, H-11, H-1, H, d, 6Hz, 6, H,6, H, 1, H, 1, 6, H, 1, H, 1, H, 1, H, 1, 6H, 6H, 6, 2, 6H, 1, 6H, 1, H, 2H, 1H, 1H, 1,2, H, 1H, 1H, 1H, 1H, 2, 1H, 2, H, 12, H, 1H, 0.79(3H, d, J ═ 6.9Hz, H-3"). 13C-NMR (cd3cl3,125mhz): δ C211.83 (C-3), 204, as shown in fig. 18.09(C-1'),198.35(C-1),168.04(C-4a),164.33(C-6),159.36(C-8),153.75(C-4b),112.27(C-9a),105.70(C-5), 103.77(C-8a),100.53(C-7),56.15(C-2),53.43(C-2'),47.47(C-4),34.79(C-1"),31.55(C-9),25.29(C-12),25.14 (C-13),24.91(C-3'),24.74(C-10),24.14(C-11),22.85(C-4'),22.68(C-5'),18.90(C-2"),18.74(C-3").

Isomethyltucommulone B, the chemical structure of which is shown in FIG. 19; the molecular formula is as follows: c₂₅H₃₂O₆. As shown in fig. 20, 1H NMR (CD3Cl3,500MHz): δ H6.14 (1H, s, H-5),4.33(1H, d, J ═ 4.3Hz, H-9),3.92(1H, m, J ═ 6.6Hz, H-2'),2.01(1H, m, J ═ 3.5 Hz, H-1"),1.39(3H, s,2-CH3),1.42(3H, s,2-CH3),1.44(3H, s,4-CH3),1.48(3H, s,4-CH3),1.23(3H, d, J ═ 6.6Hz,2' -CH3),1.21(3H, d, J ═ 6.6Hz,2' -CH3),0.80(6H, t, J ═ 6H, 2, t ═ 2 CH 357 ″, C7315 ″, C-3C 7315 ″, 3C-3H, 3-4933C 3H, 3-3H 3-3C 31, 3H 3-3H, 3-3C 31H 3-C31H 3-3H, 3H 3-C31H, 3-C31H 3-3H, C31H, C3H, C3H 3, C3H, C3H 3, C3, C3, 47.34(C-4),167.93(C-4a),95.07(C-5),156.41(C-10a), 106.93(C-7),162.15(C-8),105.01(C-9),111.79(C-9a),156.4(C-10a),211.02(C-1'),39.8(C-2'),34.52(C-1"), 31.94(2-CH3),25.12(C),24.57(4-CH3),23.94(),19.26(2'-CH3),19.09(C-),18.79 (1' -CH3).