CN112986216B - Method for detecting typical chemical components of skin-care traditional Chinese medicine product by surface enhanced Raman spectroscopy - Google Patents
Method for detecting typical chemical components of skin-care traditional Chinese medicine product by surface enhanced Raman spectroscopy Download PDFInfo
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Abstract
The invention relates to a method for detecting typical chemical components of a skin-care traditional Chinese medicine product by surface-enhanced Raman spectroscopy. The skin-care traditional Chinese medicine product is prepared from traditional Chinese medicine phellodendron bark, typical chemical components comprise berberine and possibly added dexamethasone, and the method comprises the following steps: (1) mixing the skin care traditional Chinese medicine product for detection with a saturated sodium chloride solution, dispersing a sample, adding acetonitrile, extracting, performing vortex treatment, and centrifuging to obtain a supernatant; (2) loading the supernatant onto a pre-activated extraction column for leaching, eluting with methanol, and collecting the eluate as a test solution; (3) weighing berberine hydrochloride and dexamethasone to obtain reference solution; (4) adding 50 mu l of gold nano sol into the test solution and the reference solution, shaking uniformly, measuring on a Raman spectrometer, and recording the spectrum of Raman shift; (5) reading the recorded Raman spectrum and judging the result. The method of the present invention exhibits excellent technical effects as described in the specification.
Description
Technical Field
The invention belongs to the technical field of traditional Chinese medicines, relates to a detection method of typical chemical components in a skin-care traditional Chinese medicine product, and particularly relates to a detection method of typical chemical components in a skin-care traditional Chinese medicine product containing a cortex phellodendri medicinal material. Typically, the present invention is a method for detecting typical chemical components in skin care traditional Chinese medicine products comprising traditional Chinese medicine phellodendron bark using surface enhanced raman spectroscopy.
Background
Skin disorders are very common in infants and young children, and are typically eczema, itchy skin, scarred skin, and the like. In view of the delicate skin of infants, clinically, topical Chinese medicinal preparations are generally used for symptomatic treatment of the skin diseases.
Phellodendron amurense is added in the form of extract in the prescription of a plurality of skin-care traditional Chinese medicine products suitable for infants.
The cortex Phellodendri is bark of phellodendron amurense or phellodendron chinense of Rutaceae. Collected between 3 and 6 months. Selecting more than ten years of phellodendron bark, and taking part of bark in turn. Can not be stripped off at one time to keep the original tree to continue growing. After stripping, new skin grows automatically, and the uncut part can be harvested in the next year. Drying the rest bark to semi-dry, flattening, scraping off coarse bark (cortex Coryli Heterophyllae) until it is yellow, and the endothelium is not damaged, brushing, drying in the sun, standing in dry and ventilated place, and preventing mildew and discoloration.
The phellodendron is deciduous tree and is 10-25 meters high; the outer layer of the bark is gray, the bark has a thick cork layer, the surface has longitudinal fissures, and the inner skin is bright yellow. Small branches are usually grayish brown or light brown, rarely reddish orange. The leaf pair, single feather complex leaf, 5-13 lobules, short lobule stem, long round lobule, oval, 5-11 cm long, 2-3.8 cm wide, tip length tapered, base part of wide wedge or nearly round, edge with thin round sawtooth or nearly toothless, often by margin hair; the upper side is dark green, and the vessels are soft and hairy in young and smooth and unhaired in old. The lower side is pale, the peripheral vessels are soft and hairy in young and the middle vessels are white and long and soft in old. The inflorescence is conical, and the axes and branches are shoddy; the flower is parthenocarpic, is female and male, and is small; calyx 5, oval; petal 5, long round, with yellow green; a stamen 5 of the male flower, which extends out of the petals and the base part of the filament is hairy; the degenerated stamen of the female flower is flaky, 1 part of the pistil is in an upper ovary, the style of the flower is very short, and the head of the stigma is in a head shape and is 5 parts. The berry-shaped stone fruit is spherical, the diameter of the stone fruit is about 8-10 mm, the stone fruit is purple black when the stone fruit is mature, and 5 stones are provided. The flowering period is 5-6 months. The fruit period is 9-10 months. Phellodendron bark, cortex Phellodendri, is produced in Sichuan, Guizhou, Hubei and Yunnan provinces. In addition, it is produced in Hunan, Gansu and Guangxi provinces. The yield is the largest in Sichuan and Guizhou.
Cortex Phellodendri contains alkaloids such as berberine, jateorhizine, magnoflorine, phellodendrine, N-methyl hordenine, tetrandrine, dauricine, etc.; and further contains obacunone, obacunlactone, dictamnolide, obacunone acid, green fluorescent acid, 7-dehydrostigmasterol, beta-sitosterol, and campesterol. The cortex Melaleucae Leucadendrae contains berberine, jateorhizine, phellodendrine and N-methyl hordenine. Wood also contains berberine. The fresh leaves contain phellodendrin, dehydrophellodendrin, dehydroisocoryzanin, and isocoryzanin. The dried leaves contain hyperin and no phellodendron glycosides.
Huang Bai is cold in nature and bitter in taste, entering kidney and bladder meridians. The clinical functional indications include: clearing heat, eliminating dampness, purging pathogenic fire, and removing toxic substances; it is commonly used for treating dysentery due to heat, diarrhea, diabetes, jaundice, flaccidity, 36484m, nocturnal emission, stranguria with turbid urine, hemorrhoid, hematochezia, leukorrhagia, bone steaming, overstrain and heat, conjunctival congestion, sore of mouth and tongue, and pyocutaneous disease and pyogenic infections.
The phellodendron bark contains more berberine, which greatly contributes to the pharmacological action of the phellodendron bark. The pharmacological action of phellodendron is mainly as follows: the antibacterial action: the cortex Phellodendri contains berberine as antibacterial effective component. In vitro tests show that the composition is effective on staphylococcus aureus, pneumococcus, diphtheria bacillus, viridans streptococcus, dysentery bacillus (except Sonne) and the like, almost has no effect on escherichia coli and typhoid bacillus, and reports show that the composition is effective on escherichia coli and pseudomonas aeruginosa. As for crude drugs, the antibacterial effect of coptis chinensis is 1 time stronger than that of phellodendron amurense. The compound has no direct inhibition effect on H37Rv and tubercle bacillus avium, but can reduce the number of bacteria, or shows bacteriostasis effect at a very high concentration (1/100), but reports that good results are obtained. When guinea pigs are inoculated with human tubercle bacillus for experimental treatment, the curative effect of oral administration or injection is poor. The hydrochloric acid crystal extracted from phellodendron amurense has certain curative effect on guinea pig inoculated with tubercle bacillus bovis by intramuscular injection. The phellodendron bark is said to improve the clinical symptoms and X-ray examination of tuberculosis patients and is superior to coptis chinensis. ② the function of reducing blood pressure: cortex Phellodendri can produce significant and persistent blood pressure lowering effect when injected into the vein or abdominal cavity of anesthetized animals, and carotid artery injection is stronger than intravenous injection, so that blood pressure lowering is probably central. The tertiary amine compound, namely the piroctone can also cause rapid and remarkable blood pressure reduction by changing quaternary ammonium phellodendrine; the spinal cord was cut between the I-II cervical vertebrae, and the blood pressure reduction disappeared, so that it was also proved that the blood pressure reduction is central. In addition, the Xiropirine has stronger anti-epinephrine-like effect, and can inhibit the pressure-boosting reaction caused by compressing carotid artery, asphyxiation and electrically stimulating large visceral nerve and the transient membrane contraction reaction caused by injecting epinephrine or electrically stimulating sympathetic ganglion on neck. Benzamine, tolazarin, reserpine, etc. all can reduce the hypotensive reaction. Other effects: phellodendrine or piroctone has an inhibitory effect on the central nervous system, and the spontaneous activity and various reflexes of the mouse are inhibited; given the unaesthetic rabbit with piroctone, brain waves can appear as high-amplitude slow waves. Phellodendrine has mild toxigenic effect, has no effect on tension of rectus abdominis of frog, but can inhibit contraction reaction caused by acetylcholine. For isolated rabbit intestine, the phellodendron bark powder can enhance the amplitude, and the phellodendron ketone can enhance the tension and amplitude; phellodendron lactone inhibits intestinal tract. Cortex Phellodendri has the effect of promoting pancreatic secretion in rabbits with pancreatic fistula. When the dose of phellodendron lactone is close to the lethal dose (0.05-0.1 g/kg), the phellodendron lactone can reduce the blood sugar of rabbits, and the phellodendron ketone has no effect. The phellodendron bark is said to have the function of protecting blood platelets, and is yet to be proved. It is reported that phellodendron amurense has an effect of killing wigglers (10 P.M. and 62% within 18 hours) and houseflies. In mice, it is taken together with Hemerocallis fulva root to reduce the toxicity of the latter.
Unfortunately, there are some skin-care Chinese medicine products on the market with illegal addition of hormone drugs, typically dexamethasone. Monitoring such illegal addition activities is an important responsibility of the drug testing workers. However, no existing technology can detect dexamethasone simultaneously with the typical substances in the traditional Chinese medicine.
Therefore, the skilled person still expects a method capable of accurately detecting certain chemical substances in the skin-care traditional Chinese medicine product, in particular a method for simultaneously determining berberine and possibly illegally added dexamethasone in the skin-care traditional Chinese medicine product containing the traditional Chinese medicine phellodendron bark.
Disclosure of Invention
The invention aims to provide a method for accurately detecting certain chemical substances in a skin-care traditional Chinese medicine product, in particular to a method for simultaneously detecting berberine and possibly illegally added dexamethasone in the skin-care traditional Chinese medicine product containing traditional Chinese medicine phellodendron bark. Surprisingly, the method for detecting typical chemical components in a skin-care traditional Chinese medicine product containing traditional Chinese medicine phellodendron bark by using surface enhanced raman spectroscopy can provide a new effective way for the drug detection work of the skin-care traditional Chinese medicine product, and the invention is completed based on the discovery.
In the present invention, the term "skin-care Chinese medicine product" is essentially a skin-care Chinese medicine preparation, i.e. a form of Chinese medicine product, such as ointment, cream, gel, etc.
Therefore, the invention provides a method for detecting typical chemical components of a skin-care traditional Chinese medicine product by using surface enhanced Raman spectroscopy, wherein the skin-care traditional Chinese medicine product is an external preparation product prepared from traditional Chinese medicine phellodendron bark, and the typical chemical components comprise berberine and possibly added dexamethasone.
The method according to the first aspect of the invention, comprising the steps of:
(1) mixing the skin care traditional Chinese medicine product for detection with a saturated sodium chloride solution in a weight ratio of 1:15, dispersing the sample, and then adding acetonitrile in a weight ratio of 1: 10, mixing and extracting, performing vortex treatment for 10min, and centrifuging to obtain a supernatant;
(2) loading the supernatant onto a pre-activated HLB solid-phase extraction column, sequentially eluting with 15ml of eluent A and 10ml of eluent B, eluting with 4ml of methanol, and collecting the eluent as a test solution;
(3) weighing appropriate amount of berberine hydrochloride and dexamethasone reference substance, and preparing into 1mg/ml reference substance solution with methanol;
(4) adding 50 mu l of gold nano sol into the test solution and the reference solution, shaking up, measuring on a Raman spectrometer, and recording the Raman shift at 1000-2000 cm-1(ii) a spectrum of light;
(5) reading the recorded Raman spectrum, wherein the Raman shift in the spectrum of the test solution of skin care Chinese medicinal product prepared from cortex Phellodendri is 1475 + -5 cm-1The characteristic peak of the berberine exists and is basically consistent with the characteristic peak in the berberine reference substance solution; at 1665 + -5 cm-1If the characteristic peak exists and is basically consistent with the characteristic peak in the dexamethasone reference substance solution, the skin-care traditional Chinese medicine product contains dexamethasone, and if the characteristic peak does not exist, the skin-care traditional Chinese medicine product does not contain dexamethasone.
The process according to the first aspect of the present invention, wherein the eluent a is a 10% acetonitrile aqueous solution and to which 0.02% formic acid and 0.025% sodium glycerophosphate are further added; the leacheate B is a 10% acetonitrile water solution.
According to the method of the first aspect of the invention, the skin-care traditional Chinese medicine product is prepared from typhonium giganteum, gallnut, spina gleditsiae, cortex pseudolaricis, cortex phellodendri, radix stemonae, rhizoma smilacis glabrae, radix sophorae flavescentis, fructus cnidii and auxiliary materials.
According to the method of the first aspect of the invention, the skin-care traditional Chinese medicine product is prepared from cicada slough, phellodendron amurense, fructus kochiae, cortex dictamni, mint, fructus cnidii, wild chrysanthemum flower, radix sophorae flavescentis and auxiliary materials.
According to the method of the first aspect of the invention, the skin-care traditional Chinese medicine product is prepared from phellodendron, lonicera confusa, sophora flavescens, coix seed and auxiliary materials.
The method according to the first aspect of the present invention, wherein the skin-care traditional Chinese medicine product is prepared from spina gleditsiae, typhonium giganteum, cortex pseudolaricis, cortex phellodendri, radix stemonae, gallnut, ilex rotundus, radix euphorbiae lantu, radix sophorae flavescentis and auxiliary materials.
According to the method of the first aspect of the present invention, the skin care traditional Chinese medicine product is prepared from aloe, wild chrysanthemum flower, radix sophorae flavescentis, cortex phellodendri, radix stemonae, fructus cnidii and auxiliary materials.
According to the method of the first aspect of the invention, the skin-care traditional Chinese medicine product is prepared from cortex dictamni, honeysuckle, wild chrysanthemum flower, fructus cnidii, golden cypress, fructus kochiae and auxiliary materials.
According to the method of the first aspect of the present invention, the skin-care traditional Chinese medicine product is prepared from fructus cnidii, fructus kochiae, radix sophorae flavescentis, cortex phellodendri, honeysuckle and auxiliary materials.
According to the method of the first aspect of the invention, the skin-care traditional Chinese medicine product is prepared from liquorice, phellodendron, honeysuckle, dandelion, Chinese violet and auxiliary materials.
According to the method of the first aspect of the invention, the following high performance liquid chromatography is further used for simultaneously determining the content of berberine and dexamethasone in the skin-care traditional Chinese medicine product:
chromatographic conditions and system applicability test: octadecylsilane chemically bonded silica is used as a filling agent; the method comprises the following steps of mixing methanol: 0.05% phosphoric acid solution (50:50) (0.1 g sodium heptanesulfonate per 100 ml) as mobile phase; the detection wavelength was 265 nm. The number of theoretical plates is not less than 4000 calculated according to berberine hydrochloride peak.
Preparation of control solutions: taking appropriate amount of berberine hydrochloride reference substance and dexamethasone reference substance, respectively, precisely weighing, adding mobile phase to obtain solution containing 0.1mg per 1ml, and obtaining berberine hydrochloride reference substance solution and dexamethasone reference substance solution. The two control solutions were mixed at a ratio of 1:1 to mix to make the system suitability test solution.
Preparation of a test solution: the test solution obtained in the above step (2) is used as a test solution.
The determination method comprises the following steps: precisely sucking 5 μ l of control solution, 5 μ l of system applicability test solution and 5-20 μ l of test solution, respectively, injecting into liquid chromatograph, measuring, and calculating.
In the method steps of the present invention, although the specific steps described therein differ in some detail or in language from the steps described in the examples of the detailed description section below, those skilled in the art can, nevertheless, fully generalize the above-described method steps in light of the detailed disclosure of the present invention.
Any embodiment of any aspect of the invention may be combined with other embodiments, as long as they do not contradict. Furthermore, in any embodiment of any aspect of the invention, any feature may be applicable to that feature in other embodiments, so long as they do not contradict. The invention is further described below.
All documents cited herein are incorporated by reference in their entirety and to the extent such documents do not conform to the meaning of the present invention, the present invention shall control. Further, the various terms and phrases used herein have the ordinary meaning as is known to those skilled in the art, and even though such terms and phrases are intended to be described or explained in greater detail herein, reference is made to the term and phrase as being inconsistent with the known meaning and meaning as is accorded to such meaning throughout this disclosure.
The present invention exhibits the technical effects as described in the present specification.
Detailed Description
The present invention will be further described by the following examples, however, the scope of the present invention is not limited to the following examples. It will be understood by those skilled in the art that various changes and modifications may be made to the invention without departing from the spirit and scope of the invention. The present invention has been described generally and/or specifically with respect to materials used in testing and testing methods. Although many materials and methods of operation are known in the art for the purpose of carrying out the invention, the invention is nevertheless described herein in as detail as possible.
The raman spectrometer used in the embodiment of the present invention was an OPAL3000 raman spectrometer from the metal company. Berberine is known to be 1475 +/-5 cm-1There is a typical characteristic peak and dexamethasone has no peak, and dexamethasone is 1665 + -5 cm-1The invention can be used for simply and qualitatively determining whether dexamethasone is added in the skin-care traditional Chinese medicine product containing the golden cypress or not. In the specific experiments of the present invention, gold nanosols were purchased from Xiamensis Gregorian, unless otherwise specifiedSol-type SERS enhancing reagent GP-S1 product
Example 1: skin-care traditional Chinese medicine product measured by surface enhanced Raman spectroscopy
Inspecting the product: the commercial SFC cream is prepared from typhonium giganteum, Chinese gall, spina gleditsiae, pseudolarix, phellodendron amurense, radix stemonae, rhizoma smilacis glabrae, radix sophorae flavescentis, fructus cnidii and auxiliary materials according to the description of the cream.
(1) Mixing the skin care traditional Chinese medicine product for detection with a saturated sodium chloride solution in a weight ratio of 1:15, dispersing the sample, and then adding acetonitrile in a weight ratio of 1: 10, mixing and extracting, performing vortex treatment for 10min, and centrifuging to obtain a supernatant;
(2) loading the supernatant onto a pre-activated HLB solid-phase extraction column, sequentially eluting with 15ml of eluent A and 10ml of eluent B, eluting with 4ml of methanol, and collecting the eluent as a test solution;
(3) weighing appropriate amount of berberine hydrochloride and dexamethasone reference substance, and preparing into 1mg/ml reference substance solution with methanol;
(4) adding 50 mu l of gold nano sol into the test solution and the reference solution, shaking up, measuring on a Raman spectrometer, and recording the Raman shift at 1000-2000 cm-1(ii) a spectrum of light;
(5) reading the recorded Raman spectrum, wherein the Raman shift in the spectrum of the test solution of skin care Chinese medicinal product prepared from cortex Phellodendri is 1475 + -5 cm-1The characteristic peak of the berberine exists and is basically consistent with the characteristic peak in the berberine reference substance solution; at 1665 + -5 cm-1If the characteristic peak exists and is basically consistent with the characteristic peak in the dexamethasone reference substance solution, the skin-care traditional Chinese medicine product contains dexamethasone, and if the characteristic peak does not exist, the skin-care traditional Chinese medicine product does not contain dexamethasone.
As a result: in the Raman spectrum, at 1474cm-1The characteristic peak of berberine exists (the method is reliable); at 1665 + -5 cm-1Has no characteristic peak (showing that the skin care traditional Chinese medicine product has no dexamethasone).
The eluent A used in step (2) of examples 1-8 of the present invention is 10% acetonitrile in water, and further 0.02% formic acid and 0.025% sodium glycerophosphate are added; the leacheate B used in the step (2) is a 10% acetonitrile water solution.
Example 2: skin-care traditional Chinese medicine product measured by surface enhanced Raman spectroscopy
Inspecting the product: the CW cream sold in the market is prepared from cicada slough, phellodendron, fructus kochiae, cortex dictamni, mint, fructus cnidii, wild chrysanthemum, radix sophorae flavescentis and auxiliary materials according to the description of the CW cream.
(1) Mixing the skin care traditional Chinese medicine product for detection with a saturated sodium chloride solution in a weight ratio of 1:15, dispersing the sample, and then adding acetonitrile in a weight ratio of 1: 10, mixing and extracting, performing vortex treatment for 10min, and centrifuging to obtain a supernatant;
(2) loading the supernatant onto a pre-activated HLB solid-phase extraction column, sequentially eluting with 15ml of eluent A and 10ml of eluent B, eluting with 4ml of methanol, and collecting the eluent as a test solution;
(3) weighing appropriate amount of berberine hydrochloride and dexamethasone reference substance, and preparing into 1mg/ml reference substance solution with methanol;
(4) adding 50 mu l of gold nano sol into the test solution and the reference solution, shaking uniformly, then placing the mixture on a Raman spectrometer for measurement, and recording the Raman shift at 1000-2000 cm-1(ii) a spectrum of light;
(5) reading the recorded Raman spectrum, wherein the Raman shift in the spectrum of the test solution of skin care Chinese medicinal product prepared from cortex Phellodendri is 1475 + -5 cm-1The characteristic peak of the berberine exists and is basically consistent with the characteristic peak in the berberine reference substance solution; at 1665 + -5 cm-1If the characteristic peak exists and is basically consistent with the characteristic peak in the dexamethasone reference substance solution, the skin-care traditional Chinese medicine product contains dexamethasone, and if the characteristic peak does not exist, the skin-care traditional Chinese medicine product does not contain dexamethasone.
As a result: in the Raman spectrum, at 1473cm-1The characteristic peak of berberine exists (the method is reliable); at 1665 + -5 cm-1Has no characteristic peak (showing that the skin care traditional Chinese medicine product has no dexamethasone).
Example 3: skin-care traditional Chinese medicine product measured by surface enhanced Raman spectroscopy
Inspecting the product: commercially available YETH gum is prepared from cortex Phellodendri, flos Lonicerae, radix Sophorae Flavescentis, Coicis semen and adjuvants according to the description.
(1) Mixing the skin care traditional Chinese medicine product for detection with a saturated sodium chloride solution in a weight ratio of 1:15, dispersing the sample, and then adding acetonitrile in a weight ratio of 1: 10, mixing and extracting, performing vortex treatment for 10min, and centrifuging to obtain a supernatant;
(2) loading the supernatant onto a pre-activated HLB solid-phase extraction column, sequentially eluting with 15ml of eluent A and 10ml of eluent B, eluting with 4ml of methanol, and collecting the eluent as a test solution;
(3) weighing appropriate amount of berberine hydrochloride and dexamethasone reference substance, and preparing into 1mg/ml reference substance solution with methanol;
(4) adding 50 mu l of gold nano sol into the test solution and the reference solution, shaking up, measuring on a Raman spectrometer, and recording the Raman shift at 1000-2000 cm-1(ii) a spectrum of light;
(5) reading the recorded Raman spectrum, wherein the Raman shift in the spectrum of the test solution of skin care Chinese medicinal product prepared from cortex Phellodendri is 1475 + -5 cm-1The characteristic peak of the berberine exists and is basically consistent with the characteristic peak in the berberine reference substance solution; at 1665 + -5 cm-1If the characteristic peak exists and is basically consistent with the characteristic peak in the dexamethasone reference substance solution, the skin-care traditional Chinese medicine product contains dexamethasone, and if the characteristic peak does not exist, the skin-care traditional Chinese medicine product does not contain dexamethasone.
As a result: in the Raman spectrum, at 1474cm-1The characteristic peak of berberine exists (the method is reliable); at 1665 + -5 cm-1Has no characteristic peak (showing that the skin care traditional Chinese medicine product has no dexamethasone).
Example 4: skin-care traditional Chinese medicine product measured by surface enhanced Raman spectroscopy
Inspecting the product: the commercially available FB cream is described according to its specification to be prepared from spina Gleditsiae, herba Typhonii gigantei, cortex pseudolaricis, cortex Phellodendri, radix Stemonae, Galla chinensis, radix Ilicis Pubescentis, radix Euphorbiae Fischerianae, radix Sophorae Flavescentis and adjuvants.
(1) Mixing the skin care traditional Chinese medicine product for detection with a saturated sodium chloride solution in a weight ratio of 1:15, dispersing the sample, and then adding acetonitrile in a weight ratio of 1: 10, mixing and extracting, performing vortex treatment for 10min, and centrifuging to obtain a supernatant;
(2) loading the supernatant onto a pre-activated HLB solid-phase extraction column, sequentially eluting with 15ml of eluent A and 10ml of eluent B, eluting with 4ml of methanol, and collecting the eluent as a test solution;
(3) weighing appropriate amount of berberine hydrochloride and dexamethasone reference substance, and preparing into 1mg/ml reference substance solution with methanol;
(4) adding 50 mu l of gold nano sol into the test solution and the reference solution, shaking up, measuring on a Raman spectrometer, and recording the Raman shift at 1000-2000 cm-1(ii) a spectrum of light;
(5) reading the recorded Raman spectrum, wherein the Raman shift in the spectrum of the test solution of skin care Chinese medicinal product prepared from cortex Phellodendri is 1475 + -5 cm-1The characteristic peak of the berberine exists and is basically consistent with the characteristic peak in the berberine reference substance solution; at 1665 + -5 cm-1If the characteristic peak exists and is basically consistent with the characteristic peak in the dexamethasone reference substance solution, the skin-care traditional Chinese medicine product contains dexamethasone, and if the characteristic peak does not exist, the skin-care traditional Chinese medicine product does not contain dexamethasone.
As a result: in the Raman spectrum, at 1475cm-1The characteristic peak of berberine exists (the method is reliable); at 1665 + -5 cm-1Has no characteristic peak (showing that the skin care traditional Chinese medicine product has no dexamethasone).
Example 5: skin-care traditional Chinese medicine product measured by surface enhanced Raman spectroscopy
Inspecting the product: the commercially available DMR cream is described in the specification to be made from aloe, wild chrysanthemum flower, sophora flavescens, phellodendron, stemona, cnidium fruit and auxiliary materials.
(1) Mixing the skin care traditional Chinese medicine product for detection with a saturated sodium chloride solution in a weight ratio of 1:15, dispersing the sample, and then adding acetonitrile in a weight ratio of 1: 10, mixing and extracting, performing vortex treatment for 10min, and centrifuging to obtain a supernatant;
(2) loading the supernatant onto a pre-activated HLB solid-phase extraction column, sequentially eluting with 15ml of eluent A and 10ml of eluent B, eluting with 4ml of methanol, and collecting the eluent as a test solution;
(3) weighing appropriate amount of berberine hydrochloride and dexamethasone reference substance, and preparing into 1mg/ml reference substance solution with methanol;
(4) adding 50 mu l of gold nano sol into the test solution and the reference solution, shaking up, measuring on a Raman spectrometer, and recording the Raman shift at 1000-2000 cm-1(ii) a spectrum of light;
(5) reading the recorded Raman spectrum, wherein the Raman shift in the spectrum of the test solution of skin care Chinese medicinal product prepared from cortex Phellodendri is 1475 + -5 cm-1The characteristic peak of the berberine exists and is basically consistent with the characteristic peak in the berberine reference substance solution; at 1665 + -5 cm-1If the characteristic peak exists and is basically consistent with the characteristic peak in the dexamethasone reference substance solution, the skin-care traditional Chinese medicine product contains dexamethasone, and if the characteristic peak does not exist, the skin-care traditional Chinese medicine product does not contain dexamethasone.
As a result: in the Raman spectrum, at 1474cm-1The characteristic peak of berberine exists (the method is reliable); at 1662cm-1Has a characteristic peak (showing that dexamethasone is contained in the skin-care traditional Chinese medicine product).
Example 6: skin-care traditional Chinese medicine product measured by surface enhanced Raman spectroscopy
Inspecting the product: the commercially available WF cream is described in the specification to be prepared from cortex dictamni, honeysuckle, wild chrysanthemum, fructus cnidii, phellodendron, fructus kochiae and auxiliary materials.
(1) Mixing the skin care traditional Chinese medicine product for detection with a saturated sodium chloride solution in a weight ratio of 1:15, dispersing the sample, and then adding acetonitrile in a weight ratio of 1: 10, mixing and extracting, performing vortex treatment for 10min, and centrifuging to obtain a supernatant;
(2) loading the supernatant onto a pre-activated HLB solid-phase extraction column, sequentially eluting with 15ml of eluent A and 10ml of eluent B, eluting with 4ml of methanol, and collecting the eluent as a test solution;
(3) weighing appropriate amount of berberine hydrochloride and dexamethasone reference substance, and preparing into 1mg/ml reference substance solution with methanol;
(4) adding the test solution and the reference solution50 mul of gold nano sol is shaken up and then is placed on a Raman spectrometer for measurement, and the Raman shift is recorded to be 1000-2000 cm-1(ii) a spectrum of light;
(5) reading the recorded Raman spectrum, wherein the Raman shift in the spectrum of the test solution of skin care Chinese medicinal product prepared from cortex Phellodendri is 1475 + -5 cm-1The characteristic peak of the berberine exists and is basically consistent with the characteristic peak in the berberine reference substance solution; at 1665 + -5 cm-1If the characteristic peak exists and is basically consistent with the characteristic peak in the dexamethasone reference substance solution, the skin-care traditional Chinese medicine product contains dexamethasone, and if the characteristic peak does not exist, the skin-care traditional Chinese medicine product does not contain dexamethasone.
As a result: in the Raman spectrum at 1472cm-1The characteristic peak of berberine exists (the method is reliable); at 1664cm-1Has a characteristic peak (showing that dexamethasone is contained in the skin-care traditional Chinese medicine product).
Example 7: skin-care traditional Chinese medicine product measured by surface enhanced Raman spectroscopy
Inspecting the product: the commercial AH cream is prepared from fructus cnidii, fructus kochiae, radix sophorae flavescentis, cortex phellodendri, honeysuckle and auxiliary materials according to the description of the AH cream.
(1) Mixing the skin care traditional Chinese medicine product for detection with a saturated sodium chloride solution in a weight ratio of 1:15, dispersing the sample, and then adding acetonitrile in a weight ratio of 1: 10, mixing and extracting, performing vortex treatment for 10min, and centrifuging to obtain a supernatant;
(2) loading the supernatant onto a pre-activated HLB solid-phase extraction column, sequentially eluting with 15ml of eluent A and 10ml of eluent B, eluting with 4ml of methanol, and collecting the eluent as a test solution;
(3) weighing appropriate amount of berberine hydrochloride and dexamethasone reference substance, and preparing into 1mg/ml reference substance solution with methanol;
(4) adding 50 mu l of gold nano sol into the test solution and the reference solution, shaking up, measuring on a Raman spectrometer, and recording the Raman shift at 1000-2000 cm-1(ii) a spectrum of light;
(5) reading the recorded Raman spectrum, and performing Raman shift in the spectrum of test solution of skin care Chinese medicinal product prepared from cortex PhellodendriAt 1475 +/-5 cm-1The characteristic peak of the berberine exists and is basically consistent with the characteristic peak in the berberine reference substance solution; at 1665 + -5 cm-1If the characteristic peak exists and is basically consistent with the characteristic peak in the dexamethasone reference substance solution, the skin-care traditional Chinese medicine product contains dexamethasone, and if the characteristic peak does not exist, the skin-care traditional Chinese medicine product does not contain dexamethasone.
As a result: in the Raman spectrum, at 1473cm-1The characteristic peak of berberine exists (the method is reliable); at 1663cm-1Has a characteristic peak (showing that dexamethasone is contained in the skin-care traditional Chinese medicine product).
Example 8: skin-care traditional Chinese medicine product measured by surface enhanced Raman spectroscopy
Inspecting the product: the commercially available PYS cream is described in the specification to be made from licorice, phellodendron, honeysuckle, dandelion, viola and auxiliary materials.
(1) Mixing the skin care traditional Chinese medicine product for detection with a saturated sodium chloride solution in a weight ratio of 1:15, dispersing the sample, and then adding acetonitrile in a weight ratio of 1: 10, mixing and extracting, performing vortex treatment for 10min, and centrifuging to obtain a supernatant;
(2) loading the supernatant onto a pre-activated HLB solid-phase extraction column, sequentially eluting with 15ml of eluent A and 10ml of eluent B, eluting with 4ml of methanol, and collecting the eluent as a test solution;
(3) weighing appropriate amount of berberine hydrochloride and dexamethasone reference substance, and preparing into 1mg/ml reference substance solution with methanol;
(4) adding 50 mu l of gold nano sol into the test solution and the reference solution, shaking up, measuring on a Raman spectrometer, and recording the Raman shift at 1000-2000 cm-1(ii) a spectrum of light;
(5) reading the recorded Raman spectrum, wherein the Raman shift in the spectrum of the test solution of skin care Chinese medicinal product prepared from cortex Phellodendri is 1475 + -5 cm-1The characteristic peak of the berberine exists and is basically consistent with the characteristic peak in the berberine reference substance solution; at 1665 + -5 cm-1If the characteristic peak exists and is basically consistent with the characteristic peak in the dexamethasone reference solution, the content of the ground in the skin-care traditional Chinese medicine product is shownAnd if the dexamethasone is not present, the dexamethasone is not present in the skin-care traditional Chinese medicine product.
As a result: in the Raman spectrum, at 1474cm-1The characteristic peak of berberine exists (the method is reliable); at 1662cm-1Has a characteristic peak (showing that dexamethasone is contained in the skin-care traditional Chinese medicine product).
Example 9: skin-care traditional Chinese medicine product measured by surface enhanced Raman spectroscopy
The procedure of examples 1 to 4 was followed, except that dexamethasone was added to the sample (sample: dexamethasone =10g:10mg), and the results were determined in the same manner as follows: in the Raman spectrogram, 1475 +/-5 cm-1Characteristic peaks of berberine exist in all the parts (which indicates that the method is reliable); at 1665 + -5 cm-1All have dexamethasone characteristic peak.
Example 10: skin-care traditional Chinese medicine product measured by surface enhanced Raman spectroscopy
The procedure of examples 5 to 8 was followed except that formic acid was not added to the eluent A used in the step (2), and as a result: in the Raman spectrogram, 1475 +/-5 cm-1No berberine characteristic peak is detected (indicating that the method is unreliable); at 1665 + -5 cm-1All have dexamethasone characteristic peak.
Example 11: skin-care traditional Chinese medicine product measured by surface enhanced Raman spectroscopy
The procedures of examples 5 to 8 were followed except that sodium glycerophosphate was not added to the eluent A used in the step (2), and as a result: in the Raman spectrogram, 1475 +/-5 cm-1No berberine characteristic peak is detected (indicating that the method is unreliable); at 1665 + -5 cm-1All have dexamethasone characteristic peak.
Example 12: skin-care traditional Chinese medicine product measured by surface enhanced Raman spectroscopy
The procedure of examples 5 to 8 was followed except that neither formic acid nor sodium glycerophosphate was added to the eluent A used in the step (2), and as a result: in the Raman spectrogram, 1475 +/-5 cm-1No berberine characteristic peak is detected (indicating that the method is unreliable); at 1665 + -5 cm-1All have dexamethasone characteristic peak. Based on the results of examples 10-12 and examples 5-8 above, it was unexpectedly found that step (I)2) The addition of formic acid and sodium glycerophosphate to the eluent a was necessary to retain both berberine and dexamethasone in the extraction column.
Example 13: HPLC method for measuring skin-care traditional Chinese medicine product
In the embodiment, berberine and dexamethasone in the skin-care traditional Chinese medicine product are simultaneously measured by high performance liquid chromatography.
The measurement according to the standard of the high performance liquid chromatography described in the 2020 edition of the general rules 0512 of the four departments of Chinese pharmacopoeia comprises the following operations:
chromatographic conditions and system applicability test: octadecylsilane chemically bonded silica is used as a filling agent; the method comprises the following steps of mixing methanol: 0.05% phosphoric acid solution (50:50) (0.1 g sodium heptanesulfonate per 100 ml) as mobile phase; the detection wavelength was 265 nm. The number of theoretical plates is not less than 4000 calculated according to berberine hydrochloride peak.
Preparation of control solutions: taking appropriate amount of berberine hydrochloride reference substance and dexamethasone reference substance, respectively, precisely weighing, adding mobile phase to obtain solution containing 0.1mg per 1ml, and obtaining berberine hydrochloride reference substance solution and dexamethasone reference substance solution. The two control solutions were mixed at a ratio of 1:1 to mix to make the system suitability test solution.
Preparation of a test solution: the test solution obtained in step (2) of examples 1 to 12 is used as a test solution.
The determination method comprises the following steps: precisely sucking 5 μ l of control solution, 5 μ l of system applicability test solution and 5-20 μ l of test solution, respectively, injecting into liquid chromatograph, measuring, and calculating.
In the HPLC method of this example, the retention time of berberine is about 13min, the relative retention time of dexamethasone to berberine is 1.26, and the separation degree of the two is greater than 6. In addition, in the above HPLC method, if the sodium heptanesulfonate is changed to the same amount of sodium dodecylsulfonate, the separation degree between dexamethasone and berberine is 1.82, which is obviously worse than that when sodium heptanesulfonate is used. In addition, in the above HPLC method, if methanol in the mobile phase was replaced with an equal amount of acetonitrile, the degree of separation between dexamethasone and berberine was 2.27, which is significantly worse than using methanol. The above-described HPLC method of the present invention is therefore beneficial for the effective use of berberine and dexamethasone.
The HPLC method of this example was used to measure the test solutions obtained in step (2) of examples 1 to 12, and the results were:
in the examples 1-4, berberine is detected in the test solution, and dexamethasone is not detected in the test solution;
in examples 5-8, berberine and dexamethasone were detected in the test solutions, and the peak area ratio of berberine to dexamethasone was 1: in the range of 0.21-0.36, for example, the peak area ratio of berberine to dexamethasone in the test solution of example 5 is 1: 0.274.
example 9 berberine and dexamethasone were detected in both test solutions, with the peak area ratio between them being 1: 0.17-0.34, for example, the ratio of the peak areas of berberine and dexamethasone in the test solution obtained by the method in reference example 1 is 1: 0.202.
in the examples 10-12, no berberine or only a weak chromatographic peak is detected in the test solution, and dexamethasone is detected and is equivalent to the peak areas in the examples 5-8.
The result of this example 13 shows that the reliability of the method for measuring berberine, a typical substance of phellodendron bark, and dexamethasone which may be added in skin care traditional Chinese medicine products in examples 1-12 by using raman spectroscopy is verified through HPLC method detection. HPLC methods can quantify but are cumbersome to detect, while raman spectroscopy, while it is not advantageous for quantitative measurements, can be very simple to perform qualitatively with a hand-held raman spectrometer.
The above-mentioned embodiments are merely preferred embodiments for fully illustrating the present invention, and the scope of the present invention is not limited thereto. The equivalent substitution or change made by the technical personnel in the technical field on the basis of the invention is all within the protection scope of the invention. The protection scope of the invention is subject to the claims.
Claims (9)
1. A method for detecting typical chemical components of a skin-care traditional Chinese medicine product by using surface enhanced Raman spectroscopy, wherein the skin-care traditional Chinese medicine product is prepared from traditional Chinese medicine phellodendron bark, the typical chemical components comprise berberine and possibly added dexamethasone, and the method comprises the following steps of S1 to S5:
s1: mixing the skin care traditional Chinese medicine product for detection with a saturated sodium chloride solution in a weight ratio of 1:15, dispersing the sample, and then adding acetonitrile in a weight ratio of 1: 10, mixing and extracting, performing vortex treatment for 10min, and centrifuging to obtain a supernatant;
s2: loading the supernatant onto a pre-activated HLB solid-phase extraction column, sequentially eluting with 15ml of eluent A and 10ml of eluent B, eluting with 4ml of methanol, and collecting the eluent as a test solution;
s3: weighing appropriate amount of berberine hydrochloride and dexamethasone reference substance, and preparing into 1mg/ml reference substance solution with methanol;
s4: adding 50 mu l of gold nano sol into the test solution and the reference solution, shaking up, measuring on a Raman spectrometer, and recording the Raman shift at 1000-2000 cm-1(ii) a spectrum of light;
s5: reading the recorded Raman spectrum, wherein the Raman shift in the spectrum of the test solution of skin care Chinese medicinal product prepared from cortex Phellodendri is 1475 + -5 cm-1The characteristic peak of the berberine exists and is basically consistent with the characteristic peak in the berberine reference substance solution; at 1665 + -5 cm-1If the characteristic peak exists and is basically consistent with the characteristic peak in the dexamethasone reference substance solution, the skin-care traditional Chinese medicine product contains dexamethasone, if the characteristic peak does not exist, the skin-care traditional Chinese medicine product does not contain dexamethasone,
wherein:
the leacheate A is a 10% acetonitrile aqueous solution and is added with 0.02% formic acid and 0.025% sodium glycerophosphate;
the leacheate B is a 10% acetonitrile water solution;
the skin care traditional Chinese medicine product is prepared from typhonium giganteum, gallnut, spina gleditsiae, pseudolarix, phellodendron amurense, radix stemonae, rhizoma smilacis glabrae, radix sophorae flavescentis, fructus cnidii and auxiliary materials.
2. The method of claim 1, wherein the skin care herbal product is made from periostracum Cicadae, cortex Phellodendri, Kochiae fructus, cortex Dictamni Radicis, herba Menthae, fructus Cnidii, flos Chrysanthemi Indici, radix Sophorae Flavescentis and adjuvants.
3. The method of claim 1, wherein the skin care herbal product is made from phellodendron amurense, lonicera confusa, sophora flavescens, coix lachryma-jobi kernel and adjuvants.
4. The method of claim 1, wherein the skin care herbal product is made from spina Gleditsiae, herba Typhonii gigantei, cortex pseudolaricis, cortex Phellodendri, radix Stemonae, Galla chinensis, ilex rotundus, radix Euphorbiae Fischerianae, radix Sophorae Flavescentis, and adjuvants.
5. The method of claim 1, wherein the skin care herbal product is made from aloe, wild chrysanthemum flower, sophora flavescens, phellodendron amurense, stemona, cnidium fruit and auxiliary materials.
6. The method of claim 1, wherein the skin care herbal product is made from cortex Dictamni, flos Lonicerae, flos Chrysanthemi Indici, fructus Cnidii, cortex Phellodendri, Kochiae fructus and adjuvants.
7. The method of claim 1, wherein the skin care herbal product is made from cnidium fruit, kochia fruit, sophorae flavescentis, phellodendron bark, honeysuckle flower and auxiliary materials.
8. The method of claim 1, wherein the skin care herbal product is made from licorice, phellodendron, honeysuckle, dandelion, viola yedoensis and auxiliary materials.
9. The method of claim 1, further comprising simultaneously measuring the content of berberine and dexamethasone in the skin care traditional Chinese medicine product by using high performance liquid chromatography as follows:
chromatographic conditions and system applicability test: octadecylsilane chemically bonded silica is used as a filling agent; methanol in a volume ratio of 50: 0.05 percent of phosphoric acid solution and 0.1g of sodium heptanesulfonate is added into every 100ml of the mixed solution as a mobile phase; the detection wavelength is 265 nm; the number of theoretical plates is not less than 4000 calculated according to berberine hydrochloride peak;
preparation of control solutions: respectively taking appropriate amount of berberine hydrochloride reference substance and dexamethasone reference substance, precisely weighing, adding mobile phase to obtain solution containing 0.1mg per 1ml, to obtain berberine hydrochloride reference substance solution and dexamethasone reference substance solution; the two control solutions were mixed at a ratio of 1:1, mixing and preparing a system applicability test solution according to the volume ratio;
preparation of a test solution: using the test solution obtained in step S2 of claim 1 as a test solution;
the determination method comprises the following steps: precisely sucking 5 μ l of control solution, 5 μ l of system applicability test solution and 5-20 μ l of test solution, respectively, injecting into liquid chromatograph, measuring, and calculating.
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