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Hoạt tính giảm đau và tiêu chuẩn hóa bột sấy phun được chiết xuất từ các bộ phận của cây ngải cứu Artemisia vulgaris L

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Nghiên cứu này được thực hiện với mục tiêu là chuẩn hóa bột phun sấy của A. vulgaris (SDA) bằng cách xác định các thông số hóa lý, sự hiện diện hay có mặt của kim loại nặng và một số loài vi khuẩn; sàng lọc hóa thực vật; xây dựng phương pháp sắc ký lỏng áp suất cao (HPLC) để xác định hàm lượng eupatilin; đánh giá tác dụng giảm đau trên mô hình gây đau quặn bằng acid acetic.

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Nội dung Text: Hoạt tính giảm đau và tiêu chuẩn hóa bột sấy phun được chiết xuất từ các bộ phận của cây ngải cứu Artemisia vulgaris L

TẠP CHÍ Y HỌC VIỆT NAM TẬP 539 - THÁNG 6 - SỐ CHUYẤN ĐỀ - 2024 HOẠT TÍNH GIẢM ĐAU VÀ TIÊU CHUẨN HÓA BỘT SẤY PHUN ĐƯỢC CHIẾT XUẤT TỪ CÁC BỘ PHẬN CỦA CÂY NGẢI CỨU ARTEMISIA VULGARIS L Nguyễn Văn Thư1, Trịnh Nam Trung1, Cao Vân Anh1, Phạm Đức Thịnh1, Đặng Tiến Trường1, Nguyễn Duy Bắc2 TÓM TẮT 69 được từ nghiên cứu này có thể được sử dụng để Giới thiệu: Cây Ngải cứu có tên khoa học là chuẩn hóa bột phun sấy A. vulgaris. Artemisia vulgaris L., là một loài có tầm quan trọng rất lớn trong lịch sử y học. Phương pháp SUMMARY nghiên cứu: Nghiên cứu này được thực hiện với ANALGESIC ACTIVITY AND mục tiêu là chuẩn hóa bột phun sấy của A. STANDARDIZATION OF SPRAY- vulgaris (SDA) bằng cách xác định các thông số DRIED POWDERS PREPARED FROM hóa lý, sự hiện diện hay có mặt của kim loại nặng AERIAL PARTS EXTRACTS OF và một số loài vi khuẩn; sàng lọc hóa thực vật; ARTEMISIA VULGARIS L xây dựng phương pháp sắc ký lỏng áp suất cao Introduction: Artemisia vulgaris L., (HPLC) để xác định hàm lượng eupatilin; đánh commonly known as mugwort, is a species with giá tác dụng giảm đau trên mô hình gây đau quặn great importance in the history of medicine. bằng acid acetic. Kết quả: Tỷ lệ độ ẩm, tro toàn Methods: The present investigation was carried phần, tro không tan trong aacid của SDA lần lượt out to standardize the spray-dried powders of A. là 3,95% - 4,57%, 6,59% - 7,02%, 0,004%- vulgaris (SDA) by determination of 0,006%. Nồng độ của tất cả các kim loại nặng physicochemical parameters, presence or absence được thử nghiệm đều nằm dưới giới hạn chấp of heavy metals, and microbial contamination; nhận được của WHO và các loài vi khuẩn như screening for phytochemicals; development of Escherichia coli, Salmonella spp, Staphylococcus High Pressure Liquid Chromatography (HPLC) aureus và Pseudomonas aeroginosa không có for the determination of eupatilin; and acetic acid trong SDA. Phân tích hóa thực vật cho thấy SDA induced writhing assays were employed to as có chứa flavonoid, tannin và phenolics. Hàm certain analgesic effect. Results: The lượng eupatilin là 0,89 – 0,90 mg/g. Bột phun sấy percentages of moisture content, total ash, acid có tác dụng giảm đau. Kết luận: kết quả thu insoluble ash of SDA were 3.95% - 4.57%, 6.59% - 7.02%, 0.004%-0.006%, respectively. The concentrations of all the tested heavy metals 1 Viện Đào tạo Dược, Học viện Quân y were below the WHO acceptable limits and 2 Bộ môn Giải phẫu, Học viện Quân y bacterial species, such as Escherichia coli, Chịu trách nhiệm chính: Nguyễn Văn Thư, Salmonella spp, Staphylococcus aureus, and Nguyễn Duy Bắc Pseudomonas aeroginosa were not present in Email: thu_vmmu@hotmail.com SDA. The phytochemical analysis showed that Ngày nhận bài: 16/4/2024 SDA contain flavonoids, tannins, and phenolics. Ngày phản biện khoa học: 26/4/2024 The content of eupatilin was 0.89 – 0.90 mg/g. Ngày duyệt bài: 12/5/2024 497
HỘI NGHỊ KHOA HỌC TOÀN QUỐC THƯỜNG NIÊN HỘI HÌNH THÁI HỌC VIỆT NAM - 2024 The spray-dried powders possessed analgesic nervous system disorders such as insomnia, effect. Conclusion: the results obtained from this epilepsy, depression, and excessive stress study can be used to standardize the spray-dried exposure. Furthermore, it is recommended powder of A. vulgaris. for relieving hypertension and inducing labor Keywords: Artemisia vulgaris, Asteraceae, or miscarriage. Scientific study on the plant Analgesic. shows a wide range of activities, including antioxidant, antitumor, antispasmodic, anti- I. INTRODUCTION steroidogenic, anti-fertility, antihypertensive, Pain is defined as an unpleasant sensory muscle relaxant, antiviral, antibacterial, and emotional experience associated with cholinergic, diuretic etc. Previous studies actual or potential tissue damage.1 According dealing with A. vulgaris phytochemistry to the National Institutes of Health, pain is point out the presence of different chemical one of our most important national public groups, such as essential oil, polysaccharide, health problems, a silent epidemic. For many flavonoids, hydroxycinnamic acids, and people, pain is more or less a permanent quinic acid derivatives.3,4 Thus, this study feature of their lives and has a profound aimed to investigate the potential analgesic impact on their quality of life. Currently, activity of a standardised spray dried powder opioids and non-steroidal anti-inflammatory extract of A. vulgaris in animal model. drugs (NSAIDs) are used in the treatment of pain.2 Although these drugs have excellent II. MATERIALS AND METHODS analgesic effects, however, they are they are Plant material said to have numerous side effects: opioids The aerial parts of A. vulgaris were cause physical dependency, tolerance, and collected from Phu Tho Province, Vietnam addiction while NSAIDs usually cause and identified by Dr. V.H. Do from gastrointestinal disorders.As such, research Department of Plant Resources, Institute of to discover other alternatives to treat pain is Ecology and Biological Resources, Vietnam crucial. Plants are being used in the Academy of Science and Technology. A traditional systems of medicine in many parts voucher specimen (LHT-QY001) was of the world for the control, management deposited in the Institute of Pharmaceutical and/or treatment of a variety of human and Education, Vietnam Military Medical animal ailments. Many of these herbs with University, 160 Phung Hung, Ha Dong analgesic activity had been used without any District, Hanoi, Viet Nam. adverse effects. Preparation of spray-dried powder The plant Artemisia vulgaris L., a from hydroalcoholic extract of Artemisia perennial weed, commonly known as vulgaris mugwort, and locally known in the Vietnam The extraction of A. vulgaris aerial parts as “Ngai cuu”, is distributed widely in Asia, was done using ultrasonic bath (Model 2510, Europe, Northern Africa and North Branson Ultrasonics Corporation, America.1-2 The plant is traditionally used for Connecticut, USA) designed with a fixed treatment of gynecological ailments, frequency of 40 kHz and power intensity 160 gastrointestinal diseases, cholera, leprosy W. The sample was mixed with 95 % ethanol 498
TẠP CHÍ Y HỌC VIỆT NAM TẬP 539 - THÁNG 6 - SỐ CHUYẤN ĐỀ - 2024 in the flask in the ratio of 1:15 (g/mL). The Hydrochloride acid (2 N; 25 mL) was ultrasound-assisted extraction was done at added to the crucible containing the total ash, temperature 40°C for 30 min. The extract covered with a watch glass and boiled gently was then filtered through Whatman no. 1, for 5 min. The watch glass was rinsed with 5 dried using a rotary evaporator (Buchi R220, mL of hot water and the rinsed contents were Germany) under vacuum at 40°C until added to the crucible. The acid insoluble reaching the solid content of 12%. Drying matter was collected on an ashless filter carriers (maltodextrin:aerosil, 7:1) were paper and washed with hot water until the added to the concentrated extract (8%, wet filtrate was neutral. The filter paper base) was further subjected to a Mini Spray Dryer B-290 (Buchi, Flawil, Switzerland) to containing acid insoluble matter was obtain the powder form the extract. The transferred to the original crucible, dried and operational conditions were: inlet ignited to a constant weight. The residue was temperature 140°C, outlet temperature allowed to cool in a desicator and weighed. 102°C; 4 mL/min feed rate and 2 bar The content of the acid insoluble ash in mg/g spraying pressure. of air dried material was calculated.8 Determination of physicochemical Determination of heavy metals parameters of Artemisia vulgaris spray- The contents of lead (Pb), cadmium (Cd), dried powder arsenic (As) and mercury (Hg) have been Physicochemical parameters such as detected in spray dried powder using AAS moisture content, total ash content, acid (Atomic Absorption Spectroscopy) according insoluble ash content were determined for A. to AOAC.9 vulgaris spray-dried powder according to the Determination of microbial methods described in guidelines of WHO.5 contamination in the spray dried powder Determination of moisture content Presence or absence of yeasts, moulds, The powdered material (1 g) was placed in a moisture dish and dried to a constant Escherichia coli, Salmonella spp, weight in an oven at 105°C. The loss of Staphylococcus aureus and Pseudomonas weight in mg/g of air-dried material was aeroginosa were determined according to the calculated.6 standards of TCVN.10 Determination of total ash content Preliminary phytochemical screening The powdered material (1 g) was of Artemisia vulgaris spray-dried powder accurately weighed and placed in a crucible. The qualitative chemical tests were The material was ignited to a constant weight performed for the A. vulgaris spray-dried by gradually increasing the heat to 600°C ± powder according to the methods described 25oC until it was white. The residual ash was by Farnsworth11 with some modifications. allowed to cool in a desicator. The content of Test for phenolics total ash in mg/g of air dried material was Two to three drops of 1% FeCl3 solution calculated.7 was added into 2 mL portions (1%) of the Determination of acid insoluble ash spray-dried powder. Phenolic compounds content produce a deep violet color with ferric ions. 499
HỘI NGHỊ KHOA HỌC TOÀN QUỐC THƯỜNG NIÊN HỘI HÌNH THÁI HỌC VIỆT NAM - 2024 Test for tannins were performed by comparing absorbance The spray-dried powder was dissolved in spectra and retention times with those of the methanol (1-2 mL) by heating. A few drops standard reference. The concentrations of of 5% FeCl3 solution was added. Tannins eupatilin in the samples were estimated give a blackish blue or green blackish color based on the regression line of eupatilin in in the presence of FeCl3. the range of 6.25 - 100 µg/ml, which was Y Test for flavonoids = 29927X + 84570, where Y is the peak area The spray-dried powder was dissolved in of the analyte and X is the concentration of methanol (1-2 mL) by heating. Then metal the analyte (μg/ml). magnesium and 5-6 drops of conc. HCl were Analgesic activity of A. vulgaris spray- added. The solution turns red when dried powders flavonoids are present. To evaluate whether SDA has peripheral Determination of eupatilin in spray- analgesic activity, we performed acetic acid dried powders of Artemisia vulgaris writhing test. Acetic acid-induced writhing A 500 mg portion of the extracts of SDA test was performed as reported previously.12 was dissolved in 20 ml methanol and The animals of either sex were weighed and subjected to ultrasonication for 20 min. Then divided into four groups of ten animals in the volume of the content was made up to 25 each. Control group (CG), which received ml using a volumetric flask. Finally, all the gavage with normal saline (0.1 ml/10 g); samples were passed through a 0.45 μm positive group (PG), which received PTFE filter before HPLC injection. diclofenac sodium (20 mg/kg, i.p.), a Similarly, the reference standards (eupatilin, standard analgesic drug13 ; low dosage group 5 mg) were dissolved in 5 ml methanol and (LG), which received gavage with SDA (170 then filtered. The stock solutions were used mg/kg) and high dosage group (HG), which to prepare further dilutions. Eupatilin received gavage with SDA (340 mg/kg). The concentration was determined by an Eclipse experiment lasted for 7 days. Two hours after XDB-C18, 5μm, 4.6 × 150 mm (Agilent, gavage on the seventh day, mice were USA) in a Waters high-performance liquid injected intraperitoneally with 0.2 ml of chromatography equipped with a photodiode 0.6% acetic acid, and the number of writhes array detector, quaternary pump, online of each mouse was counted starting from 5 degasser, auto sampler, automatic injector, min up to 20 min and expressed as column heater (Milford, MA, USA). The percentage protection. content of eupatilin was determined by the HPLC method described by Kim et al.,.12 The III. RESULTS AND DISCUSSION calibration curve was set up at 350 nm by Physicochemical parameters subjecting the eupatilin standard solution to Results are listed in Table 1. The the isocratic mobile phase, which consisted moisture content at 105oC of spray dried of acetonitrile/water/acetic acid (38:62:0.5, powders from 3.95% to 4.57% was obtained, v/v/v), at a flow rate of 1 ml/min at 30°C which can be considered as adequate with an injection volume of 20 μl. assuming the maximum moisture content recommended by the U.S. Pharmacopoeia13 Identification and calibration of the samples 500
TẠP CHÍ Y HỌC VIỆT NAM TẬP 539 - THÁNG 6 - SỐ CHUYẤN ĐỀ - 2024 for dry extracts of medicinal plants (≤5%). Staphylococcus aureus, Pseudomonas Moisture content of drug should be at aeruginosa. Based on the test results, in all minimum level to discourage the growth of samples there were no pathogenic bacteria bacteria, yeast or fungi during storage. Low tested. moisture content indicates the appropriate Preliminary phytochemical analysis standard, quality and stability of plant Phytochemical screening was carried out material and can be considered in future to identify the phytoconstituents present in study or application. If the drying process is the spray-dried powders. Based on the not efficient, it may lead to the degradation screening (Table 2), the compounds of phytoconstituents of the drug during contained in the spray-dried powders of A. storage.14 The total ash value of spray-dried vulgaris are flavonoid, tannin and phenolic. powders was found range from 6.59% to The eupatilin content (Table 2) ranged from 7.02%, while acid insoluble ash ranged from 0,89 mg/g to 0,90 mg/g. 0.004% to 0.006%, respectively. The ash Analgesic activity of A. vulgaris spray- values of the drug are also a significant dried powders parameter for the detection of nature of To evaluate whether or not analgesic material, adulteration, impurities, activities of SDA are through acting on authenticity of drug, quality and purity of the peripheral tissues, we performed acetic acid test writhing test. As shown in Table 3, SDA sample. The total ash value indicates the (low and high dosage) and diclofenac sodium impurities like carbonate, oxalate and significantly reduced the writhing number as silicate. The acid-insoluble ash is used to compared to control mice treated with CG estimate the amount of silica present, group (p < 0.05). The percentage of especially sand which is the indication of inhibition of writhing was 63.57%, 35.71% contamination with earthy material.14 and 37.86% in PG, LG and HG groups, Relatively less amount of these two respectively. Expectedly, positive drug parameters indicate low inorganic matter and diclofenac sodium showed significant silica were detected in spray-dried powders. analgesic activity with conspicuous decrease Heavy metal contamination testing aims in number of writhes and increased inhibition to determine the levels of metal content Hg, ratio. These data suggest that SDA may have Pb, Cd and As contained in spray-dried certain analgesic activity. powders, which is dangerous and toxic to the body. The concentrations of all the tested IV. CONCLUSION heavy metals were below the WHO In conclusion, the results obtained from acceptable limits. Microbial contamination physicochemical parameters, phytochemical reveals the impurity in medicinal plants, screening studies and development of HPLC which come from the preparation or final for the determination of eupatilin can be used products. Based on microbial contamination to standardize spray-dried powders of A. test, the results of total yeast and mold vulgaris. Analgesic activity of A. vulgaris number test spray-dried powders did not spray-dried powders by writhing induced contain yeast and mold. The identification method showed certain analgesic effect. test of pathogenic bacteria is carried out to Hence it is recommended that further detect the presence of pathogenic bacteria advance and high level work should be done including Escherichia coli, Salmonella sp, 501
HỘI NGHỊ KHOA HỌC TOÀN QUỐC THƯỜNG NIÊN HỘI HÌNH THÁI HỌC VIỆT NAM - 2024 on A. vulgaris spray-dried powders to use it 7. AOAC: Official methods of Analysis as a natural, economic and safe drug. (2000). Determination of lead, cadmium, and V. ACKNOWLEDGEMENT minerals in foods by Atomic Absorption This research was supported from Spectrophotometry (method 999.11/985.35). Ministry of National Defense (grant number Association of Official Analytical Chemists, 2019.75.057). Gaithersburg, USA. 8. Vietnamese Standard (TCVN) 8275- VI. CONFLICT OF INTEREST 2:2010 - ISO 21527-2:2008. Microbiology The authors declare no conﬂict of of food and animal feeding stuffs - interest. Horizontal method for the enumeration of yeasts and moulds - Part 2: Colony count REFERENCES technique in products with water activity less 1. Merskey H. “Pain terms: a list with than or equal to 0.95. Ministry of Science definitions and notes on usage. and Technology of the Socialist Republic Recommended by the lSDA Subcommittee of Vietnam, Hanoi, Vietnam. on Taxonomy”. Pain. 1979;6:249–252. 9. Farnsworth NR. Biological and 2. Balamurugan M, Parthasarathi K, phytochemical screening of plants. J Pharm Ranganathan LS et al. Hypothetical mode Sci 1996;55:225-76 of action of earthworm extract with 10. Kim JS, Cha KH, Kang SY, Won DH et al. hepatoprotective and antioxidant properties. in vivo gastric residence and gastroprotective J. Zhejiang Univ. Sci. B. 2008;9:141–147. effect of floating gastroretentive tablet of 3. Abiri R, Silva ALM, Mesquita LSSD, DA-9601, an extract of Artemisia asiatica, in Mesquita JWCD, Atabaki N, Almeida beagle dogs. Drug Des. Dev. Ther. EBD, et al. Towards a better understanding 2016;10:1917–1925. of Artemisia vulgaris: botany, 11. Gupta AK, Parasar D, Sagar A. et al. phytochemistry, pharmacological and Analgesic and anti-inflammatory properties biotechnological potential. Food Res Int. of gelsolin in acetic acid induced writhing, 2018;109: 403-15. https://doi.org/10.1016/ tail immersion and carrageenan-induced paw j.foodres.2018.03.072 edema in mice. PLoS ONE. 2015;10, 4. Soon L, Ng PQ, Chellian J, Madheswaran e0135558, https://doi.org/ 10.1371/journal. T, Panneerselvam J, Gupta G, et al. pone.0135558 Therapeutic potential of Artemisia vulgaris: 12. USP XXX, United States Pharmacopeia, an insight into underlying immunological United States Pharmacopeial Convention, mechanisms. J Environ Pathol Toxicol Rockville, Md, USA, 30th edition, 2007. Oncol. 2019;38:205-221. https://doi.org/10. 13. Evans WC (2005) Trease and Evans’ 1615/JEnvironPatholToxicolOncol.20190293 Pharmacognosy, 16th edn. Rajkamal Electric 97. press, Delhi, pp 516–536. 5. Quality control methods for medicinal 14. Rakholiya K, Kaneria M, Chandra S plant materials. Geneva: World Health (2016) Physicochemical and phytochemical Organization; 1998. analysis of different parts of Indian Kesar 6. Vietnamese Pharmacopoeia V, Medical Mango–a unique variety from Saurashtra Publishing House, Hanoi, Vietnam, 2017. Region of Gujarat. Pharmacogn J. 2016;8:502–506. 502

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