CN115141681A - Volatile oil, preparation method and application thereof - Google Patents

Volatile oil, preparation method and application thereof Download PDF

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CN115141681A
CN115141681A CN202210223813.1A CN202210223813A CN115141681A CN 115141681 A CN115141681 A CN 115141681A CN 202210223813 A CN202210223813 A CN 202210223813A CN 115141681 A CN115141681 A CN 115141681A
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CN115141681B (en
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刘智谋
乐雅武
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Hunan Nuoz Biological Technology Co ltd
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    • C—CHEMISTRY; METALLURGY
    • C11—ANIMAL OR VEGETABLE OILS, FATS, FATTY SUBSTANCES OR WAXES; FATTY ACIDS THEREFROM; DETERGENTS; CANDLES
    • C11B—PRODUCING, e.g. BY PRESSING RAW MATERIALS OR BY EXTRACTION FROM WASTE MATERIALS, REFINING OR PRESERVING FATS, FATTY SUBSTANCES, e.g. LANOLIN, FATTY OILS OR WAXES; ESSENTIAL OILS; PERFUMES
    • C11B9/00—Essential oils; Perfumes
    • C11B9/02—Recovery or refining of essential oils from raw materials
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
    • A01N65/00—Biocides, pest repellants or attractants, or plant growth regulators containing material from algae, lichens, bryophyta, multi-cellular fungi or plants, or extracts thereof
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
    • A01N65/00—Biocides, pest repellants or attractants, or plant growth regulators containing material from algae, lichens, bryophyta, multi-cellular fungi or plants, or extracts thereof
    • A01N65/08—Magnoliopsida [dicotyledons]
    • A01N65/10—Apiaceae or Umbelliferae [Carrot family], e.g. parsley, caraway, dill, lovage, fennel or snakebed
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
    • A61K36/18—Magnoliophyta (angiosperms)
    • A61K36/185—Magnoliopsida (dicotyledons)
    • A61K36/23—Apiaceae or Umbelliferae (Carrot family), e.g. dill, chervil, coriander or cumin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals

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Abstract

The present application relates to volatile oils, methods of preparation and uses thereof, in particular antiviral uses.

Description

Volatile oil, preparation method and application thereof
Technical Field
The present application relates to volatile oils, in particular derived from centella asiatica, methods of preparing and uses of said volatile oils, in particular antiviral uses, and antiviral methods using said volatile oils.
Background
Centella asiatica (l.) Urban is complex in chemical composition, and mainly contains triterpenes (such as asiaticoside, madecassoside, bolognan, ginsenosides, asiatic acid, etc.), flavonoids (such as quercetin, kaempferol, etc.), volatile oils, and polyalkynenes. The pharmacological activity of centella asiatica and its chemical composition are closely related.
Pharmacological studies on centella asiatica have focused on the triterpenoid component. For example, studies have shown that aqueous extracts of centella asiatica have central nervous system inhibitory effects, cognitive ability and memory enhancing effects; centella asiatica triterpenes extract has the function of promoting wound healing; asiaticoside has therapeutic effect on skin ulcer, keloid, and skin amyloidosis; the asiaticoside has antiinflammatory effect on skin; the herba Centellae water extract or asiaticoside (active component of herba Centellae water extract) has anti-gastric ulcer effect; and so on. However, there has been less pharmacological research on centella asiatica volatile oils. The asiatic pennywort herb volatile oil has the effect of resisting depression.
The extraction method of volatile oil mainly comprises steam steaming, extrusion, organic solvent extraction, ultrasonic or microwave extraction, ultrasonic and microwave synergistic extraction, etc. Steam distillation is the most common method for extracting volatile oil in modern industry, but has long time consumption and low yield of volatile oil. The extrusion method is complicated to operate and has low yield. The organic solvent extraction method can shorten the extraction time and improve the yield of the volatile oil, but volatile organic solvents are harmful to human bodies, and the obtained volatile oil is easy to have residual organic solvents, so the method has certain application limitation. The ultrasonic or microwave extraction method and the ultrasonic and microwave synergistic extraction method have the problems of low yield of volatile oil and solvent residue although the extraction process is simple.
The methods for extracting the asiatic pennywort herb volatile oil reported in the literature at present all have the problems of low yield, solvent residue and the like. For example, joshi, V.P., et al, extracted the volatile oil component of Centella asiatica in the western parts of Himalayas mountain by steam distillation in a yield of 0.03wt% (Joshi V P, kumar N, singh B, et al, chemical composition of the analytical oil of Centella asiatica (L.). From west western Himalaya, [ J ]. Natural Product Communications,2007,2 (5): 587-590.); oyedeji, O.A. et al extract the volatile oil from the whole dry Centella asiatica in Soxhlet extraction in 0.06wt.% (Oyedeji O A, afolayan A J., chemical composition and activity of the scientific oil of the scientific growing in South Africa, [ J ]. Pharmaceutical Biology,2005,43 (3): 249-252.).
Therefore, there is still a need to research and develop new uses of centella asiatica volatile oil and new methods for extracting centella asiatica volatile oil.
Disclosure of Invention
The inventors have found that the essential oils of the invention, in particular the essential oils (essential oils) derived from centella asiatica, have an antiviral effect, have the potential to treat diseases or conditions caused by viruses, or to inhibit viruses in the environment.
In one aspect, the present application provides a volatile oil of the present invention.
In another aspect, the present application provides a method for obtaining the essential oil of the invention.
In another aspect, the present application provides compositions comprising the essential oils of the invention.
In another aspect, the present application provides the use of a volatile oil or composition of the present invention for the preparation of an antiviral composition.
In another aspect, the present application relates to a method for treating a disease or disorder caused by a virus comprising administering to an individual in need thereof an effective amount of said volatile oil or a composition comprising said volatile oil.
In yet another aspect, the present application relates to a method for inhibiting or killing a virus in an environment comprising administering to the environment an effective amount of the volatile oil or a composition comprising the volatile oil.
The essential oil is defined below, and may be synthetic or, preferably, may be obtained from plants, preferably centella asiatica.
Detailed Description
Unless defined otherwise, technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs.
As used herein, the terms "essential oil" and "essential oil" are used interchangeably and mean syntheticOr a combination or mixture of two or more volatile components extracted from a plant (e.g., a flower, leaf, stem, root, or fruit). Methods for extraction include, but are not limited to, steam distillation, cold pressing, liposuction, and solvent extraction (e.g., CO) 2 Extraction method).
The components of the essential oil may be selected from, but are not limited to: trans-caryophyllene (CAS No. 87-44-5), alpha-humulene (CAS No. 6753-98-6), caryophyllin (CAS No. 1139-30-6), alpha-pinene (CAS No. 3856-25-5), beta-pinene (CAS No. 127-91-3), beta-elemene (CAS No. 515-13-9), thujoram (CAS No. 470-40-6), thujoram (CAS No. 16982-00-6), amyrin (CAS No. 25246-27-9), isobutylmyrcene (CAS No. 118-65-0), decyl acetate (CAS No. 112-17-4), thujoram (CAS No. 18431-82-8) eucalyptol (CAS No. 470-82-6), alpha-cubebene (CAS No. 17699-14-8), p-cymene (CAS No. 535-77-3), alpha-terpineol (CAS No. 98-55-5), linalool (CAS No. 78-70-6), D-camphor (CAS No. 464-49-3), verbenone (CAS No. 1196-01-6), borneol (CAS No. 507-70-0), bornyl levulinate (CAS No. 5655-61-8), geraniol (CAS No. 106-24-1), limonene (CAS No. 5989-27-5), camphene (CAS No. 79-92-5) and combinations or mixtures of any two or more thereof.
As used herein, the term "Centella asiatica" means Centella asiatica (L.) Urban, which is an Umbelliferae plant, for example, dried whole plant thereof.
The volatile oil according to the invention, which may be obtained from centella asiatica, may be obtained by steam distillation or soxhlet extraction, or preferably by combined use of CO 2 Supercritical extraction and molecular distillation technology, and is obtained from herba Centellae.
The term "subject" as used herein refers to an animal, including but not limited to primates (e.g., monkeys, chimpanzees, gorillas, and the like), rodents (e.g., rats, mice, gerbils, hamsters, ferrets, and the like), lagomorphs, porcines (e.g., pigs, piglets), equines, canines, felines, and the like; and a human.
The "environment" refers to the living environment, public places, living goods, vehicles, etc. of the individual, including, for example, the surfaces of the air or objects in contact with the individual.
The term "treatment" as used herein refers to a complete or partial cure for a disease, including but not limited to one, or a combination of two or more selected from the following: reducing or eliminating the etiology of the disease or disorder; ameliorating or eliminating pathological changes thereof; alleviating or eliminating one or more symptoms thereof; slowing or arresting its progression; lessening the severity thereof; reducing the incidence rate; reducing the recurrence thereof; and to improve prognosis thereof.
In this context, inhibiting or killing a virus includes, but is not limited to, reducing the number of viruses, reducing replication of the virus, reducing the activity or virulence of the virus, disrupting the structure of the virus (e.g., capsid or nucleic acid).
As used herein, the terms "comprising," including, "" having, "" containing, "or" involving, "and other variations thereof herein, are inclusive or open-ended and do not exclude additional unrecited elements or method steps, although not necessarily present (i.e., these terms also encompass the terms" consisting essentially of 8230;,/8230; composition, "and" consisting of \ 8230;/8230).
In a first aspect, the present application provides a volatile oil of the present invention.
In some embodiments, the essential oil comprises a combination selected from any one, or any two or three, or preferably all four of the following components:
based on the total weight of the volatile oil,
from about 25wt% to about 36wt% trans-caryophyllene;
from about 15wt% to about 26wt% alpha-humulene;
from about 12wt% to about 25wt% of caryophyllin; and
about 6wt% to about 17wt% of alpha-pinene.
In some embodiments, the volatile oil comprises any one of the following components, or a combination of any two or three, or preferably all four:
based on the total weight of the volatile oil,
from about 28wt% to about 35wt% trans-caryophyllene;
from about 18wt% to about 25wt% alpha-humulene;
from about 14wt% to about 24wt% of caryophyllin; and
about 7wt% to about 15wt% of alpha-pinene.
In some preferred embodiments, the essential oil comprises any one of, or a combination of any two or three, or preferably all four of the following components:
based on the total weight of the volatile oil,
from about 30wt% to about 34wt% (e.g., from about 31wt% to about 33 wt%) of trans-caryophyllene;
about 20wt% to about 24wt% (e.g., about 21wt% to about 23 wt%) of alpha-humulene;
about 15wt% to about 22wt% (e.g., about 15wt% to about 18 wt%) of caryophyllin; and
from about 8wt% to about 13wt% (e.g., from about 9wt% to about 12 wt%) of alpha-pinene.
In some further embodiments, the volatile oil further comprises any one of, a combination of any two of, or a combination of all three of the following components:
based on the total weight of the volatile oil,
from about 1.5% to about 5.0% by weight of beta-elemene;
about 1.5wt% to about 5.0wt% thujopside; and
about 1.5wt% to about 4.5wt% of thujene.
In some preferred embodiments, the amount of β -elemene is from about 2.5wt% to about 4.0wt%, preferably from about 2.8wt% to about 3.5wt% (e.g., from about 2.8wt% to about 3.2wt% or from about 2.9wt% to about 3.1 wt%), based on the total weight of the volatile oil.
In some preferred embodiments, the amount of thujopside is from about 2.5wt% to about 3.5wt%, preferably from about 2.8wt% to about 3.2wt% (e.g., from about 2.9wt% to about 3.1 wt%), based on the total weight of the volatile oil.
In some preferred embodiments, the amount of thujaplicin is about 2.4wt% to about 3.5wt%, preferably about 2.6wt% to about 3.2wt% (preferably about 2.6wt% to about 3.0wt% or about 2.6wt% to about 2.9 wt%), based on the total weight of the volatile oil.
In some further embodiments, the volatile oil further comprises, based on the total weight of the volatile oil, from about 1.5wt% to about 3.5wt% of amyrin; or
From about 1.5wt% to about 3.5wt% of isomyrcene;
or a combination thereof.
In some preferred embodiments, the amount of said myrcene is from about 1.8wt% to about 3.0wt%, preferably from about 2.0wt% to about 2.5wt% (e.g., from about 2.1wt% to about 2.4 wt%), based on the total weight of the volatile oil.
In some preferred embodiments, the amount of isoeugenol is about 1.8wt% to about 3.0wt%, more preferably about 2.0wt% to about 2.5wt% isoeugenol (e.g., about 2.1wt% to about 2.4 wt%), based on the total weight of the volatile oil.
In some further embodiments, the volatile oil further comprises, based on the total weight of the volatile oil, from about 1.2wt% to about 3.0wt% of decyl acetate; or
About 1.2wt% to about 3.0wt% of thujene;
or a combination thereof.
In some preferred embodiments, the amount of decyl acetate is from about 1.4wt% to about 2.4wt%, preferably from about 1.6wt% to about 2.0wt% (e.g., from about 1.7wt% to about 1.8 wt%), based on the total weight of the volatile oil.
In some preferred embodiments, the amount of cedrene is about 1.4wt% to about 2.4wt%, preferably about 1.6wt% to about 2.0wt% (e.g., about 1.6wt% to about 1.7 wt%), based on the total weight of the volatile oil.
In a further preferred embodiment, the volatile oil comprises, based on the total weight of the volatile oil,
from about 25wt% to about 32wt% (e.g., from about 31wt% to about 32 wt%) trans-caryophyllene;
about 18wt% to about 22wt% (e.g., about 21wt% to about 22 wt%) of alpha-humulene;
about 16wt% to about 22wt% (e.g., about 16wt% to about 22 wt%) of caryophyllin;
about 8wt% to about 12wt% (e.g., about 10wt% to about 11 wt%) of alpha-pinene (CAS No. 3856-25-5);
from about 2.8wt% to about 4.0wt% (e.g., from about 2.8wt% to about 3.0 wt%) beta-elemene;
about 2.8wt% to about 3.2wt% (e.g., about 2.8wt% to about 3.0 wt%) of thujopside;
about 2.6wt% to about 3.2wt% (e.g., about 2.6wt% to about 3.0 wt%) of thujaene;
from about 2.0wt% to about 2.5wt% of myrcene;
about 1.8wt% to about 2.5wt% (e.g., about 2.0wt% to about 2.5 wt%) of isoeugenol;
about 1.2wt% to about 2.0wt% (e.g., about 1.6wt% to about 2.0 wt%) of decyl acetate; and
about 1.6wt% to about 2.0wt% of cedrene.
In some further embodiments, the volatile oil further comprises, based on the total weight of the volatile oil, from about 2.0wt% to about 5.0wt% of other components.
In some embodiments, the other component is selected from eucalyptol, alpha-cubebene, p-cymene, beta-pinene, alpha-terpineol, linalool, D-camphor, verbenone, borneol, bornyl levulinate, geraniol, limonene, camphene, and combinations of any two or more thereof.
In some embodiments, the volatile oil is present in an amount sufficient to provide a desired level of volatile oil, based on the total weight of the volatile oil,
the amount of eucalyptol is from about 0.2wt% to about 0.8wt%, preferably from about 0.4wt% to about 0.7wt%, more preferably from about 0.5wt% to about 0.6wt%;
the amount of said alpha-cubebene is from about 0.3wt% to about 0.8wt%, preferably from about 0.4wt% to about 0.6wt%, more preferably from about 0.4wt% to about 0.5wt%;
the amount of p-cymene is about 0.2wt% to about 0.9wt%, preferably about 0.3wt% to about 0.6wt%, more preferably about 0.3wt% to about 0.5wt%;
the amount of β -pinene is from about 0.1wt% to about 1.9wt%, preferably from about 0.2wt% to about 1.5wt%;
the amounts of alpha-terpineol, linalool and D-camphor are each from about 0.1wt% to about 0.3wt%, preferably from about 0.2wt% to about 0.3wt%;
the amount of verbenone, borneol and bornyl levulinate are each from about 0.1wt% to about 0.3wt%, from about 0.1wt% to about 0.2wt%;
the amount of geraniol is from about 0.03wt% to about 0.10wt%, preferably from about 0.04wt% to about 0.09wt%;
the amount of limonene is about 0.03wt% to about 0.20wt%, preferably about 0.04wt% to about 0.18wt%; or
The amount of camphene is about 0.03wt% to about 0.16wt%, preferably about 0.04wt% to about 0.15wt%.
In some preferred embodiments, the volatile oil is present in an amount sufficient to provide, based on the total weight of the volatile oil,
the amount of eucalyptol is from about 0.4wt% to about 0.8wt%, preferably from about 0.5wt% to about 0.7wt%, more preferably from about 0.5wt% to about 0.6wt%;
the amount of said alpha-cubebene is from about 0.3wt% to about 0.8wt%, preferably from about 0.4wt% to about 0.6wt%, more preferably from about 0.4wt% to about 0.5wt%;
the amount of p-cymene is about 0.2wt% to about 0.6wt%, preferably about 0.3wt% to about 0.5wt%, more preferably about 0.3wt% to about 0.4wt%;
the amount of β -pinene is from about 0.1wt% to about 0.6wt%, preferably from about 0.2wt% to about 0.5wt%;
the amounts of alpha-terpineol, linalool and D-camphor are each from about 0.1wt% to about 0.3wt%, preferably from about 0.2wt% to about 0.3wt%;
the amount of verbenone, borneol and bornyl levulinate are each from about 0.1wt% to about 0.3wt%, from about 0.1wt% to about 0.2wt%; or
The amounts of geraniol, limonene and camphene are each from about 0.03wt% to about 0.06wt%, preferably from about 0.04wt% to about 0.06wt%.
In some particularly preferred embodiments, the essential oil comprises the components shown in table 1 below:
TABLE 1
Figure BDA0003538470730000051
Figure BDA0003538470730000061
In some embodiments, each of the volatile oil components described above as well as the volatile oil may be synthetic.
In some preferred embodiments, the volatile oil described above can be obtained from a plant, preferably centella asiatica (i.e., centella asiatica essential oil); even more preferably, the volatile oil comprises the components shown in table 2 or table 3 below:
TABLE 2
Figure BDA0003538470730000062
Figure BDA0003538470730000071
In a second aspect, the present application provides a method for obtaining the essential oil of the invention.
In some embodiments, the essential oil can be made available by CO in combination 2 An extraction method of supercritical extraction and molecular distillation technology comprises extracting herba Centellae. The method may comprise the steps of:
(1) Performing supercritical carbon dioxide extraction on the segmented asiatic pennywort herb stems and leaves to obtain a crude oily product;
(2) Performing first molecular distillation operation on the oily product obtained in the step (1), and collecting a first distillate to obtain a first asiatic pennywort herb essential oil part;
(3) Subjecting the residue after the first molecular distillation operation to a second molecular distillation operation under conditions different from the first molecular distillation operation, and collecting a second distillate to obtain a second centella essential oil fraction; and
(4) Combining the first and second centella essential oil fractions.
In some embodiments, step (1) comprises:
(1-i) feeding the fragmented centella asiatica stems and leaves into an extraction unit of a supercritical carbon dioxide extraction system to form a packed layer;
(1-ii) passing a supercritical carbon dioxide fluid from the bottom of the extraction device up through the packed layer at a temperature of about 35 to 60 ℃ and a pressure of about 15 to 40Mpa to extract the fragmented centella asiatica stem leaves; and
(1-iii) passing the stream obtained in step (1-ii) to a separation unit to obtain the crude oily product.
Preferably, in process (1-ii), the temperature is about 40 to 60 ℃, for example about 45 to 55 ℃ or about 50 to 55 ℃.
Preferably, in process (1-ii), the pressure is about 16 to 38MPa, for example about 18 to 36MPa or about 20 to 32MPa.
In some embodiments, the second molecular distillation operation is conducted at a different temperature, preferably a higher temperature, than the first molecular distillation operation. In some embodiments, the second molecular distillation operation is conducted at a different vacuum, preferably a lower vacuum, than the first molecular distillation operation. In other embodiments, the second molecular distillation operation is conducted at a different temperature and a different vacuum, preferably a higher temperature and a lower vacuum, than the first molecular distillation operation. Through the second molecular distillation operation, the yield of the centella essential oil is further improved.
In some embodiments, the first molecular distillation operation is conducted under a vacuum of about 80 to 170Pa and at a temperature of about 80 to 120 ℃. Preferably, the vacuum is about 100 to 160Pa, for example about 120 to 140Pa. Preferably, the temperature is about 90 to 120 ℃, for example about 100 to 110 ℃.
In some embodiments, the second molecular distillation operation is conducted under a vacuum of about 3 to 15Pa and at a temperature of about 150 to 200 ℃. Preferably, the vacuum is about 3 to 12Pa, for example about 5 to 8Pa or about 6 to 10Pa. Preferably, the temperature is about 160 to 190 ℃, e.g., about 170 to 180 ℃.
Optionally, each of steps (2) and (3) comprises dehydrating the distillate, or step (4) comprises dehydrating the combined distillate. The dehydration may be performed using a desiccant. Desiccants that may be utilized include, for example, anhydrous sodium sulfate, anhydrous calcium sulfate, anhydrous magnesium sulfate, and anhydrous calcium chloride. After dehydration, the drying agent may be removed by filtration to give the volatile oil.
In some embodiments, the yield of the volatile oil is about 0.08 to 0.10wt% based on the weight of the fragmented centella asiatica stems and leaves in step (1).
The inventors have also found that the oily product obtained by supercritical carbon dioxide extraction in step (1) has a high viscosity, is jelly-like and has poor fluidity. The volatile oil obtained by the molecular distillation operation has proper viscosity and good fluidity, and is smooth in flow and uniform in flow rate. This good flowability provides advantages in the formulation of, for example, volatile oils.
In some embodiments, the method further comprises the steps of:
(pre-1) drying fresh leaves and stems of centella asiatica in a drying device by circulating heating with hot air at a temperature of about 35 to 45 ℃ to obtain dried leaves and stems of centella asiatica; and
(pre-2) fragmenting the dried centella asiatica stem leaves.
The inventor finds that the problems of the existing centella essential oil extraction method are greatly related to the method for collecting and processing the centella raw material, so that a plurality of improvements are made on the two aspects.
Generally, the method of the present application utilizes only the stem and leaf portions of centella asiatica, rather than the whole plant, as it is mainly contained in the leaves of centella asiatica, thus avoiding the mixing of soil with the roots into the raw material.
In the prior art, the raw materials are generally dried by directly exposing the collected raw materials to the sun. However, this can result in loss of some of the volatile oil during drying or loss of material due to mildew on the material when exposed to rainy weather. According to the method, fresh stems and leaves of centella are dried by circulating heating at 35-45 ℃ in a drying device, so that dried stems and leaves of centella are obtained. The drying treatment has the advantage of relatively constant temperature, and the volatile oil cannot be lost.
In some embodiments, in step (pre-1), the temperature of the hot air is constant.
In some embodiments, in step (pre-1), the thickness of the stack of fresh centella asiatica stems and leaves is about 1 to 3cm. The excessively small thickness is not beneficial to fully utilizing heat energy, and the energy utilization rate is reduced; too high thickness is not beneficial to drying raw materials, and drying efficiency is reduced. Preferably, the stacking thickness is no more than about 2 centimeters. In some embodiments, in step (pre-1), the heating is for a time of about 3 to 8 hours, such as 4 to 6 hours or 5 to 7 hours.
Furthermore, the inventors segmented the dried centella asiatica stem leaves to facilitate the use of supercritical CO 2 The density of the raw materials of the centella asiatica is improved in the extraction, the feeding amount is increased, and the exposed area is increased, so that the extraction efficiency is improved.
In some embodiments, the fragmented centella asiatica stems and leaves are fragments having an average length of about 1 to 6cm, preferably about 3 to 5 cm.
The residual medicine dregs after the step (3) can be used for extracting other components in the centella so as to realize the comprehensive utilization of the raw materials and be beneficial to obviously reducing the cost.
In a third aspect, the present application provides a volatile oil obtainable by the process for obtaining a volatile oil of the present invention as described above.
In a fourth aspect, the present application provides a composition comprising the essential oil of the invention as described above, and optionally one or more physiologically or pharmaceutically acceptable additives, carriers and/or excipients.
The inventors have surprisingly found that the essential oils of the invention (such as the centella essential oil) have excellent antiviral activity.
Thus, in a fifth aspect, the present application provides an essential oil of the invention as described above, or a composition according to the fourth aspect, for use against a virus.
In a sixth aspect, the present application also provides the use of a volatile oil of the invention, or a composition according to the fourth aspect, as described above, for the preparation of an antiviral composition.
In a seventh aspect, the present application also provides a volatile oil of the invention, as described above, or a composition according to the fourth aspect, for use in the preparation of an antiviral composition.
In an embodiment according to each of the above fifth, sixth and seventh aspects, the virus is an RNA virus, in particular a single stranded RNA virus, e.g. a picornavirus. In some further embodiments, the virus is a (+) single stranded RNA virus, including (+) single stranded small RNA virus. In yet further embodiments, the virus is a (-) single stranded RNA virus.
In some preferred embodiments, the virus is selected from the group consisting of Orthomyxoviridae (Orthomyxoviridae), paramyxoviridae (Paramyxoviridae), rubella viridae (Matonaviridae), togaviridae (Togaviridae), picornaviridae (Picomaviridae), coronaviridae (Coronaviridae), and combinations thereof.
In some more preferred embodiments, the virus is selected from the group consisting of Influenza virus (Influenza virus), human parainfluenza virus (Human parainfluenza virus), rhinovirus (rhinovirus), pneumovirus (pneumvirus), rubella virus (Rubivirus), enterovirus (enterovirus), and Coronavirus (Coronavirus).
In some more preferred embodiments, the virus is selected from the group consisting of: influenza a viruses (inflenza a viruses) (including H1N1, H5N2, H7N1, H7N2, H7N3, H7N7, H7N9, H9N2 and H10N8 viruses a), influenza B viruses (inflenza B viruses), influenza C viruses (inflenza C viruses), influenza D viruses (inflenza D viruses), human parainfluenza viruses of types I, II, III and IV (HPIV-1, HPIV-2, HPIV-3, HPIV-4), respiratory syncytial viruses (Respiratory synthetic viruses), rubella viruses (Rubella viruses), coxsackie viruses (Coxsackie viruses) (including class a (CVA) and class B (CVB), particularly cvovb 3), enteroviruses (enteroviruses 68) (72-71), and combinations thereof.
In some more preferred embodiments, the virus is selected from the group consisting of: H1N1 virus a, influenza B virus, respiratory syncytial virus, rubella virus, coxsackie virus B3 (CVB 3), enterovirus type 71 (EV 71)), and combinations thereof.
In other embodiments, the virus is a DNA virus, particularly a double-stranded DNA virus. In some preferred embodiments, the virus is selected from herpes viruses (herpes viruses), such as Herpes Simplex Virus (HSV) and Varicella Zoster Virus (VZV). In some more preferred embodiments, the virus is VZV-3.
The volatile oil, the composition comprising the volatile oil, or the antiviral composition may be used for medical uses, for example, in some embodiments, as a pharmaceutical composition for preventing or treating a disease or disorder caused by any one or more of the viruses described above, in a subject in need thereof.
The disease or condition may be selected from: influenza, upper respiratory infection, respiratory syncytial virus pneumonia, rubella, gastrointestinal type cold, acute gastroenteritis, hand-foot-and-mouth disease, herpangina, non-paralytic poliomyelitis, viral meningitis, viral encephalitis, viral myocarditis, viral pericarditis, epidemic myalgia or chest pain, acute limb paralysis (acute flaccid paralysis), acute hemorrhagic conjunctivitis (acute hemorrhagic conjunctivitis), muscle stiffness (myosonic jerk), varicella, herpes zoster, severe acute respiratory syndrome, and middle east respiratory syndrome.
The pharmaceutical composition may be administered by inhalation, topically, transdermally or orally. An "effective amount" of the pharmaceutical composition may be about 0.1-5.0ml of the volatile oil per single dose, e.g., about 0.3-4.9ml, about 0.5-4.7ml, about 1.0-4.5ml, about 1.5-4.0ml, about 2.0-3.5ml, or about 2.5-3.0ml, based on the volume of the volatile oil. The effective amount will generally depend on a variety of factors including the sex, age, weight, general health of the individual being treated, the severity of the disorder or condition, the rate of administration and the judgment of the prescribing physician and can, therefore, vary. The pharmaceutical composition is for once, twice, three or more daily administrations.
In other embodiments, the volatile oil, the composition according to the fourth aspect, or the antiviral composition is for inhibiting or killing the virus in an environment, e.g. for use as a disinfectant. For this purpose, an "effective amount" of the volatile oil, the composition according to the fourth aspect, or the antiviral composition may be the amount described above, or may be a higher or lower amount, depending on factors of the environment, such as the size of the space, the degree of confinement, etc. The antiviral composition is for once, twice, three or more daily administrations, or for continuous administrations.
The antiviral compositions, including the pharmaceutical compositions, may further comprise one or more physiologically or pharmaceutically acceptable additives, carriers, and/or excipients, such as flavoring agents, preservatives, inclusion agents (e.g., cyclodextrin), propellants, and combinations thereof.
The composition according to the fourth aspect, or the antiviral composition (including the pharmaceutical composition), may be in solid, semi-solid, liquid or gaseous form, for example in the form of a spray, aerosol, fragrance, fumigant, sachet, syrup, elixir or soft capsule.
In an eighth aspect, the present application also provides a method for treating a disease or condition caused by a virus, comprising administering to an individual in need thereof an effective amount of the volatile oil as described above, the composition according to the fourth aspect, or the antiviral composition. The virus and the disease or disorder are as described above.
In a ninth aspect, the present application also provides a method for inhibiting or killing a virus in an environment comprising administering to the environment an effective amount of the volatile oil as described above, the composition according to the fourth aspect, or the antiviral composition. The virus is as described above.
Advantageous effects
The inventors have surprisingly found that the essential oils of the invention (such as the centella essential oil) have excellent antiviral activity. The method for obtaining the volatile oil of the present invention has various advantages such as more stable process, higher extraction efficiency and yield, no organic solvent residue, higher safety and cost-effectiveness, compared to known methods such as steam distillation or soxhlet extraction. Furthermore, the essential oils of the invention (for example centella essential oil) have a suitable viscosity and good flow properties.
Examples
The present invention is illustrated in more detail below by way of examples, which are not intended to limit the invention in any way, but rather the scope of the invention is defined only by the appended claims.
Example 1:
supercritical carbon dioxide extraction
10kg of centella asiatica stem and leaf segments with average length of about 3-5cm are put into an extraction tank of a supercritical carbon dioxide extraction system, carbon dioxide enters the extraction tank from the bottom at 45-50 ℃ and under the pressure of 15-36Mpa, and the carbon dioxide penetrates through a filling layer upwards to extract particles for 2 hours. The resulting carbon dioxide fluid was fed to a knockout drum where 0.09kg of an oily product was obtained at the bottom of the knockout drum, which had a higher viscosity, was jelly-like and had poor flowability.
Molecular distillation
The oily product was pumped to an evaporator and distilled twice. The first molecular distillation is carried out under the vacuum degree of 120Pa and the temperature of 90 ℃, and the obtained distillate is collected; the second molecular distillation is carried out under the vacuum degree of 3-6 Pa and the temperature of 170 ℃, and the obtained distillate is collected. The resulting distillates were combined, 0.5wt% of food grade anhydrous sodium sulfate was added, stirred for 10 minutes and allowed to stand for 5 hours, then filtered to give 0.0082kg of centella essential oil, which had good fluidity, smooth flow, and uniform flow rate.
Analysis of composition
The centella asiatica essential oil was analyzed by GC-MS (gas chromatography-mass spectrometry) and found to have the composition shown in table 3:
TABLE 3
Figure BDA0003538470730000111
Figure BDA0003538470730000121
Pinene: in the process of GC-MS analysis, the mixture of the alpha-pinene and the beta-pinene which are not separated is obtained.
Example 2
Supercritical carbon dioxide extraction
10kg of centella asiatica stem and leaf segments with average length of about 3-5cm are put into an extraction tank of a supercritical carbon dioxide extraction system, carbon dioxide enters the extraction tank from the bottom at the temperature of 50-56 ℃ and the temperature of 20-30Mpa, and upward permeates a filling layer to extract particles for 2 hours. The obtained carbon dioxide fluid enters a separation tank, and 0.097kg of oily product is obtained at the bottom of the separation tank, and the oily product has high viscosity, is jelly-like and has poor fluidity.
Molecular distillation
The oily product was pumped to an evaporator and distilled twice. The first molecular distillation is carried out under the vacuum degree of 140Pa and the temperature of 100 ℃, and the obtained distillate is collected; the second molecular distillation is carried out under the vacuum degree of 5-8 Pa and the temperature of 180 ℃, and the obtained distillate is collected. The resulting distillates were combined, 0.5wt% of food grade anhydrous sodium sulfate was added, stirred for 10 minutes and allowed to stand for 5 hours, then filtered to give 0.0095kg of centella essential oil, which had good fluidity, smooth flow, and uniform flow rate.
The centella essential oil had a composition similar to that of the centella essential oil prepared in example 1.
Example 3
Supercritical carbon dioxide extraction
10kg of centella asiatica stem and leaf segments with average length of about 3-5cm are put into an extraction tank of a supercritical carbon dioxide extraction system, carbon dioxide enters the extraction tank from the bottom at the temperature of 55-60 ℃ and the pressure of 30-40Mpa, and upward permeates a filling layer to extract particles for 2 hours. The resulting carbon dioxide fluid was fed to a knockout drum to give 0.092kg of an oily product at the bottom of the knockout drum which had a higher viscosity, was jelly-like and had poor flow properties.
Molecular distillation
The oily product was pumped into an evaporator and distilled twice. Performing the first molecular distillation at vacuum degree of 160Pa and temperature of 120 deg.C, and collecting the obtained distillate; the second molecular distillation is carried out under vacuum degree of 7-10Pa and temperature of 190 deg.C, and the obtained distillate is collected. The resulting distillates were combined, 0.5wt% of food grade anhydrous sodium sulfate was added, stirred for 10 minutes and allowed to stand for 5 hours, and then filtered to give 0.0089kg of centella essential oil, which had good fluidity, smooth flow, and uniform flow rate.
The centella essential oil had a composition similar to that of the centella essential oil prepared in example 1.
Example 4: extracting centella asiatica oil by steam distillation
100kg of dried whole centella asiatica is put into a distillation retort. Extracting by introducing steam (the steam temperature is 100-120 ℃) from the bottom of the distillation retort for 8-10 hours to obtain mixed steam. The mixed vapor was condensed by a condenser, the resulting oil-water mixture was passed through an oil-water separator, and the separated oil layer was dehydrated (0.5 wt% of food-grade anhydrous sodium sulfate) to obtain 0.032kg of centella asiatica oil.
Example 5: extracting centella asiatica oil by steam distillation
100kg of dried fragments of centella asiatica stems and leaves having an average length of 3-5cm was put into a retort. Extracting by introducing steam (the steam temperature is 100-120 ℃) from the bottom of the distillation retort for 8-10 hours to obtain mixed steam. The mixed vapor was condensed by a condenser, the resulting oil-water mixture was passed through an oil-water separator, and the separated oil layer was dehydrated (0.5 wt% food grade anhydrous sodium sulfate) to obtain 0.068kg of centella asiatica oil, the composition of which is shown in table 4:
TABLE 4
Figure BDA0003538470730000131
Pinene: in the GC-MS analysis process, a mixture of the alpha-pinene and the beta-pinene which is not separated is obtained.
Example 6: antiviral study of centella asiatica essential oil
The toxicity of centella asiatica essential oil on 4 cells (MDCK, HEp-2, vero and LLC-MK 2) for culturing virus is determined by MTT method, and antiviral test is carried out by designing proper concentration. In vitro antiviral pharmacodynamic test is carried out by adopting cytopathogenic method (CPE method) to evaluate the effect of centella essential oil on viruses causing gastrointestinal cold, skin ulcer and other diseases.
1. Test method
1.1 cell culture
Vero, canine kidney (MDCK), human larynx epidermoid carcinoma cells (HEp-2) and rhesus monkey kidney cells (LLC-MK 2) (4 cells all from Wuhan Protechs Life technologies, inc.) were cultured in DMEM medium (HyClone) containing 10% FBS (Sciencell, USA), and when the growth state was good, passaging was performed at 2-3 d intervals. Discarding the medium in the clean bench, washing 2-3 times with 1 × PBS, then adding an appropriate amount of 0.25% Trypsin-EDTA for digestion, after about 2-5 min, after cell shedding, adding an appropriate amount of DMEM medium containing 10% FBS to terminate the digestion of pancreatin, blowing into a single cell suspension, transferring into an EP tube, and centrifuging at 1000rpm for 5min. Discard medium, add fresh medium to resuspend, about 10 5 The cell density of/mL was inoculated into a new culture flask, placed at 37 ℃ and 5% 2 Culturing in an incubator.
1.2 amplification of viruses
1.2.1 influenza A virus (H1N 1): amplification of chick embryos
H1N1 strain (number: A/PR/8, as gift from Guangzhou respiratory disease institute) was inoculated into allantoic cavities of 9-day-old chick embryos for passage amplification. The method comprises the following specific steps: irradiating a 9-day-old SPF chick embryo, drawing a gas chamber and the position of the embryo, disinfecting the center of the gas chamber or the edge of the gas chamber far away from the side wall of the embryo by using a 75% alcohol cotton ball, punching a small hole in the center or the side edge of the gas chamber by using a steel cone, then vertically or slightly obliquely inserting a syringe needle into the gas chamber along the hole, entering allantoic sac, injecting 0.1-0.3 mL of virus liquid, pulling out the needle, sealing the hole by using paraffin, culturing at 35 ℃ for 48 hours, collecting the virus, marking, and storing at-80 ℃ for a medium-short term or a liquid nitrogen for a medium-long term for later use.
1.2.2 Influenza B Virus (IBV): MDCK cell expansion
Inoculating MDCK cells into a culture bottle, removing part of culture medium when the cell density reaches 70-80%, covering the rest cells, adding an appropriate amount of IBV (B/Guangdong/01.05SZSBYB7-O/2018, as a gift from Wuhan virus research institute) respectively, after the viruses are adsorbed on the cell surface (about 3h, gently shaking the culture plate every 30min to ensure uniform virus adsorption), replacing the FBS-free fresh culture medium, placing the FBS-free fresh culture medium at 35 ℃ and 5 ℃ for CO 2 Culturing in a humidified constant-temperature incubator. Observing that the cells start to generate lesions until the cells do not generate lesions (generally 2-3 days), adopting a repeated freeze thawing method, separating at 3000rpm for 10min to remove cell residues, collecting supernate, subpackaging in a freezing storage tube, labeling, and then storing at-80 ℃ for a medium-short period or in liquid nitrogen for a long period for later use.
1.2.3 Respiratory Syncytial Virus (RSV): HEp-2 cell expansion
Inoculating HEp-2 cells into culture flask, removing part of culture medium when cell density reaches 70-80%, covering the rest cells, adding appropriate amount of RSV (ATCC-VR-26 PQ), changing fresh medium without FBS after virus is adsorbed on cell surface (about 3 hr, shaking the culture plate gently every 30min to make virus adsorb uniformly), placing at 37 deg.C and 5% CO 2 Culturing in a humidified constant-temperature incubator. Observing that the cells start to generate lesions until the cells do not generate lesions (generally 3-7 days), adopting a repeated freeze thawing method, separating at 3000rpm for 10min to remove cell residues, collecting supernate, subpackaging in a freezing storage tube, labeling, and then storing at-80 ℃ for a medium-short term or in liquid nitrogen for a long term for later use.
1.2.4 rubella virus (RuV): vero cell expansion
Inoculating Vero cells into a culture flask, removing part of the culture medium when the cell density reaches 70-80%, covering the remaining cells, adding an appropriate amount of RuV virus (ATCC-VR-553), changing the fresh medium without FBS after adsorbing the virus onto the cell surface (about 3h, gently shaking the plate every 30min to make the virus adsorb uniformly), placing at 37 deg.C and 5% CO 2 Culturing in a humidified constant-temperature incubator. Observing the cells to start generating lesions until no lesion is generated (generally 7 days), adopting a repeated freeze thawing method, separating at 3000rpm for 10min to remove cell residues, collecting supernate, subpackaging in a freezing storage tube, labeling, and then storing at-80 ℃ for a medium-short period or in liquid nitrogen for a long period for later use.
1.2.5 varicella zoster virus (VZV-3): vero cell expansion
Inoculating Vero cells into a culture bottle, removing part of the culture medium when the cell density reaches 70-80%, covering the rest cells, adding a proper amount of VZV-3 virus (ATCC-VR-1433), after the virus is adsorbed on the cell surface (about 3h, gently shaking the culture plate every 30min to make the virus adsorbed uniformly), replacing the fresh culture medium without FBS, placing at 37 ℃, and 5% of CO 2 Culturing in a humidified constant-temperature incubator. When the cells were observed to start to produce lesions until they were no longer produced lesions (generally 5 to 7 days), the cells were collected by digestion with 0.25% trypsin-EDTA, resuspended in a non-serum cell culture solution, labeled and stored at-80 ℃ for a short or medium period of time or in liquid nitrogen for a long period of time.
1.2.6 Coxsackie virus (CVB 3): LLC-MK2 cell expansion
The LLC-MK2 cells are inoculated in a culture bottle, when the cell density reaches 80-90%, part of culture medium is removed, the rest cells are covered, a proper amount of CVB3 virus is added, after the virus is adsorbed on the cell surface (about 3h, the culture plate is slightly shaken every 30min to ensure that the virus is adsorbed uniformly), the virus growth culture medium is added to stop adsorption. At 37 ℃ C, 5% CO 2 Culturing in a humidified constant-temperature incubator. Observing cell to generate pathological changes till no pathological changes are generated (generally 1-4 days), repeatedly freezing and thawing at 3000rpm for 10min to remove cell residues, collecting supernatantThe solution is subpackaged in a freezing storage tube, and is stored at minus 80 ℃ for a medium-short period or in liquid nitrogen for a long period for later use after labeling.
1.2.7 enterovirus (EV 71): LLC-MK2 cell expansion
Inoculating LLC-MK2 cells into culture flask, removing part of culture medium when cell density reaches 70-80%, adding appropriate amount of EV71 (ATCC-VR-1432), and humidifying at 37 deg.C for 5% CO 2 Adsorbing for 1-2 hours in the environment, and adding a virus growth culture medium to terminate the adsorption. Observing that the cells start to generate lesions until the cells do not generate lesions (generally 2-6 days), adopting a repeated freeze thawing method, separating at 3000rpm for 10min to remove cell residues, collecting supernate, subpackaging in a freezing storage tube, labeling, and then storing at-80 ℃ for a medium-short period or in liquid nitrogen for a long period for later use.
1.3 Virus half Tissue culture infection concentration (TCID) 50 ) The virus solution collected at 1.2.1 to 1.2.7 was subjected to TCID 50 The determination of (1):
inoculating 100 μ L of corresponding cell suspension with appropriate density into 96-well cell culture plate, culturing for 24 hr, sucking out culture solution, adding 100 μ L of virus solution diluted with cell maintenance solution (virus is subjected to 10 times of virus culture) -1 、10 -2 、10 -3 、10 -4 、10 -5 、10 -6 Gradient dilution) of 10 replicate wells per dilution at 37 ℃ and 5% CO 2 Adsorbing and culturing for 3h in an incubator, adsorbing unadsorbed virus liquid, supplementing 100 mu L of cell growth maintenance liquid to each hole, and continuing culturing. Observing cytopathic effect day by day under an inverted microscope (CPE; the CPE caused by virus in cultured cells is characterized by cell rounding, stronger refractivity, fused cell protrusion, partial wall separation, cytoplasm with filamentous protrusion or pseudo-podiform, and irregular map in the whole shape to form large and round fused multinuclear giant cells). The number of wells with CPE was recorded, ending with the highest dilution at which no more lesions appeared, and the extent of cytopathic effect was expressed as "- + + + + + + +": no cytopathy "-", < 25% cytopathy "+",25% -50% cytopathy '++, 50% -75% cytopathy' ++++,>75% cytopathic "+++". According toCalculation of TCID by Reed-Muench formula 50 ：
TCID 50 = Log (dilution of virus with CPE less than 50%) + distance ratio × dilution spacing
Wherein the distance ratio = (percentage higher than 50% percent-50) ÷ (percentage higher than 50% percent-percentage lower than 50%).
1.4 concentration design of test substances
1.4.1 cytotoxicity assays
Preparing a series of concentrations of centella asiatica essential oil (concentration gradient of 5, 2.5, 1.25, 0.62, 0.31, 0.16 μ L/mL) in DMEM medium by using the centella asiatica essential oil prepared in example 1; oseltamivir (Oseltamivir phosphate granules, yichang Toguang Yangjiang pharmaceutical Co., ltd., lot: 0371912115) (concentration gradient of 20, 10, 5, 2.5, 1.25, 0.625. Mu.g/mL), ribavirin (ribavirin spray, penlanokang pharmaceutical Co., ltd., lot: 200218) (concentration gradient of 20, 10, 5, 2.5, 1.25, 0.625. Mu.g/mL), acyclovir (Shandong Fangling pharmaceutical Co., ltd., lot: 2007121) (concentration gradient of 20, 10, 5, 2.5, 1.25, 0.625. Mu.g/mL) were also prepared in series with DMEM medium. Adding the test substances to cultured MDCK, HEp-2, vero, LLC-MK2 cells, respectively, at 37 deg.C and 5% CO 2 Culturing in humidified incubator for 72h, adding MTT, culturing for 4h, measuring OD value of each well at 492nm wavelength, and calculating EC of centella asiatica essential oil on each cell 50 。
1.4.2 when the centella asiatica essential oil is toxic to cells, setting 5 test concentrations by adopting 1/2IC50 as the highest concentration and diluting downwards at 2-time intervals; when centella essential oil is not toxic to cells, 5 concentrations tested were set using 5 μ L/mL as the highest concentration, diluted down at 2-fold intervals.
1.5 detection of antiviral Effect
1.5.1 cell seeding: 24h before virus infection, corresponding cell strains which grow well are used for controlling the appropriate density (about 10) 5 Pieces/well) were inoculated uniformly into 96-well plates (100. Mu.L/well) at 37 ℃ with 5% CO 2 Culturing in an incubator;
1.5.2 when the cell density reaches 70-8 percentAfter about 0% or so, part of the medium was aspirated, the remaining just covered cells (to allow better virus adsorption to the cells), and 100 TCIDs were inoculated 50 After culturing the virus solution (50. Mu.L/well) in an incubator for 3 hours, the medium in a 96-well plate was aspirated, and test substance solutions (200. Mu.L/well) of different concentrations prepared using a serum-free cell maintenance solution (according to the cytotoxicity setting) were added to the culture solution, and the concentration of CO was 5% at 35 ℃ to 2 Culturing in an incubator;
1.5.3 grouping: normal control group: group not infected with virus; model control group: a virus-infected group; positive control group: infection group + commercial control drug, in which oseltamivir was used in the H1N1 and IBV assays; ribavirin was used for rubella virus RuV, RSV, CVB3 and EV71 trials; acyclovir was used in the VZV-3 assay; centella essential oil: infection group + centella asiatica essential oil at different concentrations.
1.5.4 evaluation of antiviral Activity (CPE method)
Cytopathic effect was observed day by day, and the number of cytopathic and non-cytopathic wells was recorded for each concentration, with continuous observation that the cytopathic effect did not increase.
1.6 evaluation of results
The cell lesion rate inhibition rate (%) = (1-each group of cells non-diseased well/8) × 100% based on the normal control group cell non-diseased rate as 100%.
Statistics of the test data were performed with the software SPSS 16.0.
2. Test results
2.1 Effect on cell proliferation
As shown in Table 5, the centella asiatica essential oil has different degrees of toxicity to MDCK cells and Vero cells within the tested concentration range (0.16-5 mu L/mL), and the IC of the centella asiatica essential oil is IC 50 5.736 mu L/mL and 1.864 mu L/mL respectively, has certain cytotoxicity to LLC-MK2, the maximum cytotoxicity is lower than 50%, and the IC is 50 >5 mu L/mL, no obvious cytotoxicity to HEp-2 cells. The highest concentration of centella asiatica essential oil in antiviral studies was set at 1 μ L/mL.
Oseltamivir, ribavirin and acyclovir have no obvious cytotoxicity to MDCK, HEp-2, vero and LLC-MK2 in the tested concentration range (0.625-20 mug/mL), so the highest concentration in antiviral research is set as 20 mug/mL.
TABLE 5 Effect of centella asiatica essential oil on proliferation of individual cells
Figure BDA0003538470730000171
2.2 Virus Titer detection
Half virus infectivity TCID of H1N1, IBV, RSV, ruV, VZV-3, CVB3 and EV71 50 Are respectively 10 -4.05 /0.1mL、10 -2.05 /0.1mL、10 -5.30 /0.1mL、10 -4.22 /0.1mL、10 -3.05 /0.1mL、10 -3 . 21 /0.1mL、10 -4.65 0.1mL, 8.91X 10 virus preparations respectively 5 8.91X 10 times of 3 5.01X 10 times of 6 6.03X 10 times of 4 8.91X 10 times of 4 6.17X 10 times of 4 Double, 2.24X 10 5 When diluted twice, 0.1mL of inoculated cells can cause lesion of 50% of cells. Take 100 TCIDs 50 The virus amount was 8910-fold, 89-fold, 50100-fold, 603-fold, 891-fold, 617-fold, and 2240-fold diluted respectively for in vitro antiviral tests.
2.3 Effect on cell viability after infection with Virus
The centella essential oil has inhibitory effects on 7 strains of viruses at different degrees, and can improve the survival ability of cells after infecting the cells at different degrees. EC for H1N1, IBV, RSV, ruV, VZV-3, CVB3 and EV71 50 0.350. Mu.L/mL, 0.412. Mu.L/mL, 0.594. Mu.L/mL, 0.431. Mu.L/mL, 0.503. Mu.L/mL, 0.422. Mu.L/mL and 0.467. Mu.L/mL, respectively.
The positive control drugs have different degrees of inhibition on 7 strains of viruses and have EC on H1N1, IBV, RSV, ruV, VZV-3, CVB3 and EV71 50 2.454. Mu.g/mL, 6.437. Mu.g/mL, 4.117. Mu.g/mL, 6.412. Mu.g/mL, 4.269. Mu.g/mL, 3.973. Mu.g/mL, and 4.021. Mu.g/mL, respectively.
TABLE 6 Effect of centella asiatica essential oil on H1N1 infection of MDCK cells
Figure BDA0003538470730000181
TABLE 7 Effect of centella asiatica essential oil on IBV infection of MDCK cells
Figure BDA0003538470730000182
TABLE 8 Effect of centella asiatica essential oil on RSV infection of HEp-2 cells
Figure BDA0003538470730000183
TABLE 9 Effect of centella asiatica essential oil on RuV infection of Vero cells
Figure BDA0003538470730000184
TABLE 10 Effect of centella asiatica essential oil on VZV-3 infection of Vero cells
Figure BDA0003538470730000191
TABLE 11 Effect of centella asiatica essential oil on CVB3 infection of LLC-MK2 cells
Figure BDA0003538470730000192
TABLE 12 Effect of centella asiatica essential oil on EV71 infection of LLC-MK2 cells
Figure BDA0003538470730000193
The results show that the centella essential oil (for example, the dosage of 0.03-1 mu L/mL) has different degrees of inhibitory action on various viruses and can protect cells from virus infection.
Various modifications of the invention in addition to those described herein will be apparent to those skilled in the art from the foregoing description. Such modifications are also intended to fall within the scope of the appended claims. Each reference, including all patents, patent applications, journal articles, books, and any other publications, cited in this application is hereby incorporated by reference in its entirety.

Claims (14)

1. A volatile oil, wherein the volatile oil comprises the following components:
based on the total weight of the volatile oil,
from about 25wt% to about 36wt%, preferably from about 28wt% to about 35wt%, more preferably from about 30wt% to about 34wt% of trans-caryophyllene;
from about 15wt% to about 26wt%, preferably from about 18wt% to about 25wt%, more preferably from about 20wt% to about 24wt% alpha-humulene;
from about 12wt% to about 25wt%, preferably from about 14wt% to about 24wt%, more preferably from about 15wt% to about 22wt% (e.g., from about 15wt% to about 18 wt%) of caryophyllin; and
from about 6wt% to about 17wt%, preferably from about 7wt% to about 15wt%, more preferably from about 8wt% to about 13wt% (e.g., 9wt% to about 12 wt%) of alpha-pinene.
2. The volatile oil of claim 1 wherein the volatile oil further comprises any one of, a combination of any two of, or a combination of all three of the following components:
based on the total weight of the volatile oil,
from about 1.5wt% to about 5.0wt%, preferably from about 2.5wt% to about 4.0wt%, more preferably from about 2.8wt% to about 3.5wt% (e.g., from about 2.8wt% to about 3.2 wt%) of beta-elemene;
about 1.5wt% to about 5.0wt%, preferably about 2.5wt% to about 3.5wt%, more preferably about 2.8wt% to about 3.2wt% of thujopside; and
about 1.5wt% to about 4.5wt%, preferably about 2.4wt% to about 3.5wt%, more preferably about 2.6wt% to about 3.2wt% (e.g., 2.6wt% to about 3.0 wt%) of thujaene;
further, the essential oil further comprises:
based on the total weight of the volatile oil,
from about 1.5wt% to about 3.5wt%, preferably from about 1.8wt% to about 3.0wt%, more preferably from about 2.0wt% to about 2.5wt% of amyrin; or
From about 1.5wt% to about 3.5wt%, preferably from about 1.8wt% to about 3.0wt%, more preferably from about 2.0wt% to about 2.5wt% of isomyrcene;
or a combination thereof;
still further, the volatile oil further comprises any one of the following components, or a combination of the two:
based on the total weight of the volatile oil,
about 1.2wt% to about 3.0wt%, preferably about 1.4wt% to about 2.4wt%, more preferably about 1.6wt% to about 2.0wt% of decyl acetate; or
About 1.2wt% to about 3.0wt%, preferably about 1.4wt% to about 2.4wt%, more preferably about 1.6wt% to about 2.0wt% of cedrene;
or a combination thereof.
3. The volatile oil of claim 1 or 2, wherein the volatile oil comprises, based on the total weight of the volatile oil,
from about 25wt% to about 32wt% (e.g., from about 31wt% to about 32 wt%) of trans-caryophyllene;
about 18wt% to about 22wt% (e.g., about 21wt% to about 22 wt%) of alpha-humulene;
about 16wt% to about 22wt% (e.g., about 16wt% to about 22 wt%) of a caryophyllin;
from about 8wt% to about 12wt% (e.g., from about 10wt% to about 11 wt%) of alpha-pinene;
from about 2.8wt% to about 4.0wt% (e.g., from about 2.8wt% to about 3.0 wt%) beta-elemene;
about 2.8wt% to about 3.2wt% (e.g., about 2.8wt% to about 3.0 wt%) of thujopside;
about 2.6wt% to about 3.2wt% (e.g., about 2.6wt% to about 3.0 wt%) of thujaene;
from about 2.0wt% to about 2.5wt% of myrcene;
about 1.8wt% to about 2.5wt% (e.g., about 2.0wt% to about 2.5 wt%) of isoeugenol;
about 1.2wt% to about 2.0wt% (e.g., about 1.6wt% to about 2.0 wt%) of decyl acetate; and
about 1.6wt% to about 2.0wt% of cedrene.
4. The volatile oil of any one of claims 1 to 3 wherein the volatile oil further comprises:
from about 2.0wt% to about 5.0wt% of other components, based on the total weight of the volatile oil;
in particular, the other component is selected from eucalyptol, alpha-cubebene, p-cymene, beta-pinene (CAS No. 127-91-3), alpha-terpineol, linalool, D-camphor, verbenone, borneol, bornyl levulinate, geraniol, limonene, camphene, and combinations of any two or more thereof;
more particularly, the volatile oil is present in an amount, based on the total weight of the volatile oil,
the amount of eucalyptol is from about 0.2wt% to about 0.8wt%, preferably from about 0.4wt% to about 0.7wt%, more preferably from about 0.5wt% to about 0.6wt%;
the amount of said alpha-cubebene is from about 0.3wt% to about 0.8wt%, preferably from about 0.4wt% to about 0.6wt%, more preferably from about 0.4wt% to about 0.5wt%;
the amount of p-cymene is about 0.2wt% to about 0.9wt%, preferably about 0.3wt% to about 0.6wt%, more preferably about 0.3wt% to about 0.5wt%;
the amount of β -pinene is from about 0.1wt% to about 1.9wt%, preferably from about 0.2wt% to about 1.5wt%;
the amounts of alpha-terpineol, linalool and D-camphor are each from about 0.1wt% to about 0.3wt%, preferably from about 0.2wt% to about 0.3wt%;
the amount of verbenone, borneol and bornyl levulinate are each from about 0.1wt% to about 0.3wt%, from about 0.1wt% to about 0.2wt%;
the amount of geraniol is from about 0.03wt% to about 0.10wt%, preferably from about 0.04wt% to about 0.09wt%;
the amount of limonene is about 0.03wt% to about 0.20wt%, preferably about 0.04wt% to about 0.18wt%;
the amount of camphene is about 0.03wt% to about 0.16wt%, preferably about 0.04wt% to about 0.15wt%.
5. The essential oil of any one of claims 1 to 4, wherein the essential oil comprises the components shown in Table 1.
6. The volatile oil of any one of claims 1 to 5, wherein the volatile oil may be synthetic or preferably obtainable from a plant, preferably centella asiatica; even more preferably, the volatile oil comprises the components shown in table 2.
7. A process for obtaining essential oils, wherein the process comprises the steps of:
(1) Performing supercritical carbon dioxide extraction on the segmented asiatic pennywort herb stems and leaves to obtain a crude oily product;
(2) Performing first molecular distillation operation on the oily product obtained in the step (1), and collecting a first distillate to obtain a first asiatic pennywort herb essential oil part;
(3) Subjecting the residue after the first molecular distillation operation to a second molecular distillation operation under conditions different from the first molecular distillation operation, and collecting a second distillate to obtain a second centella essential oil fraction; and
(4) Combining said first and second centella asiatica essential oil fractions;
specifically, step (1) includes the following processes:
(1-i) feeding the fragmented centella asiatica stems and leaves into an extraction unit of a supercritical carbon dioxide extraction system to form a packed layer;
(1-ii) passing a supercritical carbon dioxide fluid from the bottom of the extraction device up through the packed layer at a temperature of about 35 to 60 ℃ and a pressure of about 15 to 40Mpa to extract the fragmented centella asiatica stem leaves; and
(1-iii) passing the stream obtained in step (1-ii) to a separation unit to obtain the crude oily product;
more particularly, said first molecular distillation operation is carried out under a vacuum of about 80 to 170Pa and at a temperature of about 80 to 120 ℃; and/or
Said second molecular distillation operation being carried out under a vacuum of about 3 to 15Pa and at a temperature of about 150 to 200 ℃; and/or
The yield of centella essential oil is about 0.08 to 0.10wt% based on the weight of the fragmented centella asiatica stem and leaves in step (1); and/or
The fragmented centella asiatica stems and leaves are fragments having an average length of about 1 to 6cm, preferably about 3 to 5 cm.
8. The method of claim 7, wherein the method further comprises the steps of:
(pre-1) drying fresh leaves and stems of centella asiatica in a drying device by circulating heating with hot air at a temperature of about 35 to 45 ℃ to obtain dried leaves and stems of centella asiatica; and
(pre-2) fragmenting said dried centella asiatica stem leaves;
in particular, in step (pre-1), the thickness of the stack of fresh leaves of centella asiatica is between about 1 and 3cm, preferably not more than about 2cm; and/or
In step (pre-1), heating is continued for a period of about 3 to 8 hours.
9. Essential oil obtainable by the process of claim 7 or 8.
10. A composition comprising the volatile oil of any one of claims 1 to 6 and 9, and optionally one or more physiologically or pharmaceutically acceptable additives, carriers and/or excipients.
11. Use of the volatile oil of any one of claims 1 to 6 and 9 or the composition of claim 10 for the preparation of an antiviral composition;
in particular, the virus is an RNA virus, in particular a single-stranded RNA virus, such as a small RNA virus;
preferably, the virus is a (+) single stranded RNA virus, including a (+) small single stranded RNA virus; or (-) single stranded RNA virus;
preferably, the virus is selected from the group consisting of Orthomyxoviridae (Orthomyxoviridae), paramyxoviridae (Paramyxoviridae), rubella viridae (Matonaviridae), togaviridae (Togaviridae), picornaviridae (Picomaviridae), coronaviridae (Coronaviridae), and combinations thereof;
preferably, the virus is selected from the group consisting of Influenza virus (Influenza virus), human parainfluenza virus (Human parainfluenza virus), rhinovirus (rhinovirus), pneumovirus (Pneumovirus), rubella virus (Rubivirus), enterovirus (enterovirus), and Coronavirus (Coronavirus);
more preferably, the virus is selected from: influenza a virus (inflenza a virus) (including H1N1, H5N2, H7N1, H7N2, H7N3, H7N7, H7N9, H9N2 and H10N8 viruses a), influenza B virus (inflenza B virus), influenza c virus (inflenza cvvirus), influenza D virus (inflenza D virus), human parainfluenza viruses of types I, II, III and IV (HPIV-1, HPIV-2, HPIV-3, HPIV-4), respiratory syncytial virus (respiratory syncytial virus), rubella virus (Rubella virus), coxsackie virus (Coxsackie virus) (including class a (CVA) and class B (CVB), particularly cvovb 3), enterovirus (Enterovirus 68) (particularly Enterovirus 71) (71) and combinations thereof;
or alternatively
The virus is a DNA virus, in particular a double-stranded DNA virus;
preferably, the virus is selected from adenovirus (adenoviruses), herpes virus (herpes virus), such as herpes simplex virus (herpes simplex virus) and varicella zoster virus (variella-zoster virus, in particular VZV-3).
12. The use of claim 11, wherein the antiviral composition is used as a pharmaceutical composition for preventing or treating a disease or condition caused by any one or more viruses of claim 11;
in particular, the disease or condition is selected from: influenza, upper respiratory infection, respiratory syncytial virus pneumonia, rubella, gastrointestinal type cold, acute gastroenteritis, hand-foot-and-mouth disease, herpangina, nonparalytic poliomyelitis, viral meningitis, viral encephalitis, viral myocarditis, viral pericarditis, epidemic myalgia or chest pain, acute limb paralysis (acute flaccid paralysis), acute hemorrhagic conjunctivitis (acute hemorrhagic conjunctivitis), muscle stiffness (myosonic jerk), varicella, herpes zoster, skin ulcer, severe acute respiratory syndrome and middle east respiratory syndrome.
13. The use of claim 11, wherein the antiviral composition is used to inhibit or kill the virus in an environment.
14. A method for inhibiting or killing a virus in an environment comprising administering to the environment an effective amount of a volatile oil as defined in any one of claims 1 to 6 and 9 or a composition of claim 10.
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