INTRODUCTION:

The introduction of the principles of evidence-based medicine into current practice has provided a scientific rationale for the use of herbal medicines¹. The modern pharmaceutical industry is experiencing an obvious boom^2-4, but nature remains the main supplier of pharmacy raw materials. The value of natural resources lies in their diversity and unique combination of active ingredients⁵. However, the current level of scientific knowledge about certain herbs varies considerably. The ubiquitous medicinal plants are well studied and are a popular material for pharmaceutical, cosmetic and food production⁶. The situation is different with rare or obscure herbs with a limited range⁷. The difficulty of obtaining raw materials, the fact that they grow in places that are difficult to access and the lack of reliable scientific information make it difficult to study certain medicinal plants⁸. This includes the Ferula asafoetida L.

For thousands of years Ferula asafoetida L. has been used as a spice and medicinal plant. Treatises on ancient medicine have described the medicinal uses of resin and assa-foetida gums. However, the source of the resins was kept secret for a long time. In his monograph, it was not until the 1970s that scientists came to a consensus that the medicinal assa-foetida in ancient times was derived from the Iranian endemic Ferula asafoetida L., a plant with a strong garlic odour⁹. Resin and gum with similar properties and chemical composition are also extracted from a number of other species of the genus Ferula asafoetida L., which belongs to the tribe Peucedan subfamily Apioideae Drude family Apiaceae Lindl (Umbrellas). Data on the widespread distribution of ferulas in the eastern Mediterranean and Central Asia. It is found in Italy, Turkey, Iran, Afghanistan, China, Kazakhstan, Tajikistan, Uzbekistan, Turkmenistan and India on loess and loess-rubbly plains, fixed sands, sagebrush, saxaul and saltwort deserts¹⁰.

Ferula asafoetida L. is a plant with a pungent garlic odour, is about 1-1.5 m tall and has an underground part consisting of a strong root with root hairs and a stem (caudex). The morphology of the plant as follows: leaves are rosette ternate, 15cm long and 5cm wide, the upper part of the leaf is glabrous and the lower part is soft. Ferula is a perennial monocarpic ephemerid that dies off after a single flowering. Consequently, it can be harvested after the fruit has formed without affecting the population¹¹. The accumulation of resins and gum occurs during the plant’s short growing season, specifically over 2-3 spring months each year, in the leaves, stem, caudex, and root. To obtain the raw material, the root is partially dug out by hand and an incision is made. This releases a milky sap that hardens in the air and forms latex. The latex is scraped off, dried, and stored in a cool place. The procedure is repeated for 5–10 days until the sap stops oozing¹².

A review reported that the latex contains 9-65% resin, 12-48% gum, and 5-20% essential oil. The resin includes ferulic acid, vanillin, and sesquiterpene coumarins such as farnesiferols A, B, and C, umbelliprenin, umbelliferone, assafoetidin, saradaferin, assafoetidinol A and B, asacoumarin, and other sesquiterpenes. The essential oil contains numerous organic polysulphides, which are responsible for the unpleasant garlic-like smell of the plant¹³.

Of interest are compounds with antioxidant, antimicrobial and anti-inflammatory activity: ferulic acid, luteolin, vanillin and sesquiterpene coumarins. Ferulic acid (4-hydroxy-3-methoxycinnamic acid) belongs to a group of phenolic acids commonly found in plant tissues (whole grains, spinach, parsley, grapes, rhubarb and the seeds of cereals such as wheat, oats, rye and barley). Ferulic acid is described as an excellent antioxidant with low toxicity. Sufficient data have been accumulated on the antimicrobial activity (AMA) of coumarins¹⁴. The antimycobacterial properties of coumarins have also been investigated¹⁵, and a detailed review of the antimicrobial activity of plant extracts is available¹⁶.

The results suggested the possibility of external application of a combination of plant antioxidants and coumarins, which are part of the plant material Ferula asafoetida L., for the treatment of wounds. The aim of this study was to investigate the efficacy of Ferula asafoetida extract (EFV) for wound healing in an experiment.

MATERIALS AND METHODS:

At the preparatory stage of the study the plant material Ferula asafoetida L. was harvested at the Department of Pharmaceutical, Toxicological Chemistry, Pharmacognosy and Botany of the School of Pharmacy of Asfendiyarov Kazakh National Medical University. The raw material was collected from the fields of Arys district of Turkestan region of the Republic of Kazakhstan. The samples were dried, then extracted three times with methanol followed by filtration and concentrated using a rotary evaporator to obtain a solid extract for 24 h. The finished extract was used to prepare a 4% ointment on Vaseline. The reference preparation used was 100% Centella asiatica (ECA) extract (gotu kola) from Elizavecca Milky Piggy, which was also used to make a 4% petroleum jelly ointment.

Adult male mongrel rats (200-250 g) were kept at room temperature under standard vivarium conditions with free access to feed and water at a 12-hour light/dark cycle for at least 7 days before and during the experiment. All procedures were conducted in accordance with the protocol No 12(103) of October 28, 2020, approved by the Local Ethical Committee of Asfendiyarov Kazakh National Medical University (Registration number in the Republican Center for Health Development of the Ministry of Health of the Republic of Kazakhstan – LEK-I/2010-01-012).

Allogeneic fibroblast culture was isolated from the skin of new-born rats. Fibroblasts were cultured in Igla medium (Biolot, Russia) with the addition of 10% bovine embryo serum in an atmosphere of 5% CO2 at 37ºC for 6 days. After that, 20 µg/ml of Centella asiatica or Ferula asafoetida L. extract was added to the culture vials and cultivation was continued for another 48 hours. In the collection phase, total phenolic compounds and flavonoid concentrations in the studied extracts were determined according to the method described in the literature. The amount of phenols was examined by Folin-Ciocalteu method and the result was calculated in gallic acid equivalents (GAE). The flavonoid content was determined by complexation reaction with aluminium chloride and then expressed in quercetin equivalents¹⁷.

Stable 1.1-diphenyl-2-picrylhydrazyl radical (DPPH) was used to determine the free radical scavenging activity of the extract. Ascorbic acid was used as a standard. Antimicrobial activity was investigated by agar diffusion using a bacterial suspension on physiological solution (0.5 McFarland turbidity standard units) of standard strains of Staphylococcus aureus and Pseudomonas aeruginosa. Three wells were made on a dish containing the microorganisms, one of which was filled with EFV, the second with ECA and the third with a 1% methanol solution, which served as a control. AMA results were evaluated by the diameter of bacterial growth around the well in mm. The determination of the MIC was based on a sensitivity test with micro-dilutions in broth. To obtain final concentration values as high as 0 to 1000 µg/ml of extract, dilutions were prepared in 96-well microplates. A microplate reader (Multiskan Ascent, UK) was used to determine the bacterial growth rate. The proliferative activity of fibroblasts was assessed using the MTT test with yellow tetrazolium dye.

All experimental animals were anaesthetised intraperitoneally with ketamine and xylazine hydrochloride before the wound simulation procedure. Two series of experiments were carried out in the observation phase. In series A, the wound process was simulated in 3 groups of 7 animals each. The hair on the dorsal interscapular region of the animals was shaved and the skin was treated with 70% ethanol. Full-layer circular excision of the area was carried out using a sterile scalpel to simulate a 20 mm diameter wound. In group 1A (control), the wound healed naturally. In Group 2A, the wound was covered with a dressing containing Centella asiatica. Group 3A was covered with a dressing with Ferula asafoetida. The dressings were changed once a day.

In series B, a trophic ulcer was simulated in 3 groups of 6 animals by excising the rat skin on the dorsal interscapular region up to fascia superficialis in a circle 20 mm in diameter, after which the wound edges were fixed with a silicone ring with an external diameter of 20 mm using dermofacial nodal sutures. Fibroblast transplantation was then performed in all animals. Untreated fibroblasts were transplanted in group 1B. In Group 2B, fibroblasts treated with ECA were transplanted. In group 3B, fibroblasts treated with EFV were transplanted. The wound healing rate was assessed by measuring the area of the wound with a scale on a slide.

STATISTICA 10.0 software was used for statistical processing. The mean and standard deviation (M±SD) were determined for each quantitative indicator. Mann-Whitney test was used to assess the reliability of changes between the groups. The level of statistical significance was p<0.05.

RESULTS:

Latex and whole plants were used to prepare a plant extract of Ferula asafoetida L. Latex was harvested at the site of raw material collection over a fortnight in May, according to the method described in the literature¹². Whole plants were dug after fruit formation in July. All the material was dried and mixed before starting the extraction procedure. The resulting solid extract was then lyophilised in a yield of 14.3%

The study of phytochemical characteristics of Ferula asafoetida extract revealed a relatively high concentration of total phenols in the amount of 110.4±5.72 mg GAE/g of extract (GAE are gallic acid equivalents) according to the standard curve (y = 0.0046X, r2 = 0.988). The concentration of total flavonoids was 31.2±2.26 mg QE/g extract (QE – quercitin equivalents) when calculated according to the standard curve (y=0.0073X, r2=0.991). The method of least squares was used to construct the calibration curves. The determination of total phenols and flavonoids in the comparison extract was carried out. A high polyphenol concentration of 216±8.54 mg GAE/g of the extract was found. The obtained value of total flavonoids in ECA was approximately the same as in EFV, corresponding to 40±1.94 mg QE/g extract.

A study of pharmacological properties confirmed the moderate antioxidant capacity (AOC) of Ferula asafoetida L. Thus, it took 31.23±2.14 µg/ml of the extract for 50% radical inhibition of 2.2-diphenyl-1-picrylhydrazyl (DPPH) in the reaction mixture. This is 3 times higher compared to ascorbic acid AOS. Under the same conditions, the IC50 for ascorbic acid was 10.41 µg/ml. The antiradical properties of Septella asiatica extract were measured. Binding of 50% of DPPH radicals in the reaction mixture occurred by an amount of 2.33±0.44 µg/ml of ECA. Consequently, ECA proved to be 5 times more effective as an antioxidant compared to vitamin C, and 15 times more effective compared to EFV.

In a study of the antimicrobial activity of Ferula asafoetida L. in Petri dishes with a standard culture, the extract was added to wells with a diameter of 5mm. The result was considered positive if the diameter of the inhibition zone was 7mm or more. After 24hours of incubation at 37℃, the diameter of the inhibition zone for the aerobic Gram-negative culture Pseudomonas aeruginosa was 13mm. For the anaerobic gram-positive strain Staphylococcus aureus – was 19mm. The AMA of Centella asiatica was studied along the same lines. In the case of Pseudomonas bacillus, the diameter of the inhibition zone was 14 mm and in the case of Staphylococcus aureus, 7 mm.

By reading the optical density of the wells after incubation of a standard culture of the microorganism with different concentrations of EFV, the MIC values for Ferula asafoetida L. In the case of Staphylococcus aureus, the MIC for the test extract was 220 µg/ml. For Pseudomonas aeruginosa the MIC was 350 µg/ml. The study of Centella asiatica using the method described above did not reveal a MIC for Staphylococcus aureus. The MIC value of Centella asiatica extract for Pseudomonas aeruginosa was determined to be 320 µg/ml, which correlated well with the value of the minimum inhibitory concentration of EFV for this strain.

When modelling the wound surface in both series A and B, incised wounds were obtained in the experimental animals. They were characterised by smooth edges and a smooth wound surface. Moderate bleeding was observed immediately after application, but it stopped within half an hour. After anaesthesia had ended, the rats showed no pronounced pain sensation. Wound surface area in experimental animals was measured on days 0, 7, 14 and 21 for the entire time until complete healing. In experiment series A complete healing of the wound in the control group occurred on day 25. In series of experiments B – on the 18th day. No lethal cases were recorded. The average wound area on day 0 of the experiment in both series A and B was 319±6 mm (Table 1).

In experiment Series A, wound healing occurred by secondary tension. On day 7, the bottom of the wound surface was filled with granulation tissue in all animals. A crust was observed on the surface of the wound. On day 14, the appearance of the wounds differed significantly between the groups. In the group of animals in which the wound healed naturally, epithelialisation was observed, but not very pronounced. At the edges of the wound in group 2A and 3A rats, which were dressed with 4% Vaseline ointment with plant extract, there was a pronounced epithelialization, and islands of epithelium appeared in the middle. There were no significant differences between the groups in the use of ECA or EFV to prepare the ointment. Group 1A showed a similar pattern on day 19, whereas Group 2A and 3A showed complete healing of the wound on day 20 at the same time. On day 21, Group 1A still had a small, incompletely epithelialized area at the base of the wound.

In experiment Series B, the edges of the wound were fixed with a silicone ring to prevent spontaneous epithelialisation and to create favourable conditions for the application of fibroblast culture. Cell cultures after the addition of plant extracts were transplanted to the wound surface to study the effects of the active ingredients of Ferula asafoetida L. in vivo. Transplantation was performed on the 3rd day, the seeding density of fibroblasts was 1-2x10⁴ per cm² of the culture vial surface. This period was chosen to wait until the inflammatory phase of the wound injury was over, when the concentration of biologically active substances, histamine, cytokines, chemoattractants increased sharply in the lesion nidus. Four days after transplantation, on the 7th day of the wound process, a significantly more pronounced formation of granulation tissue was detected in groups 2B and 3B compared to group 1B. The area of wound closure in the groups of rats transplanted with the cell culture treated with the plant extract was twice as large compared to the group of animals transplanted with the untreated culture. On the 14th day of the experiment the observed trend in the course of the wound process between the groups persisted. The percentage of the wound closure was approximately 1.2 times less in the group of the rats transplanted with untreated fibroblasts than in the groups 2B and 3B animals. Significant wound epithelization and a clear reduction of the damaged surface area were observed in all experimental animals. The necrotic layer was clearly reduced, with mature granulation tissue adjacent to its edges.

Complete closure of the wound and the silicone ring and scab fall off due to the eruption of the sutures with the consequent formation of a scar on day 17 in Group 2B and 3B, and one day later in Group 1B. It is also worth noting that on the 7th day in groups 2A, 3A and 1B the processes of wound filling with a dark maroon scab had a similar character. The data obtained indicate the high biological activity of ointments prepared with herbal extracts of Ferula asafoetida and Centella asiatica. The in vitro metabolic activity of fibroblasts 48 hours after the addition of the plant extracts was studied by the colorimetric method after the addition of tetrazolium yellow salt. The conversion of the dye into insoluble dark blue formazan crystals is only possible in living, intact cells by mitochondrial succinate dehydrogenase. Cell culture viability was measured on days 6, 7 and 8. After that, the obtained fibroblast cultures were transplanted to experimental animals. ECA and EFV were injected on day 6. The data obtained revealed a significantly higher metabolic activity in cells treated with the plant material Ferula asafoetida L. compared to the culture treated with Centella asiatica extract (Figure 1).

Figure 1. MTT test for assessing fibroblast viability after treatment with extracts of Ferula asafoetida L. and Centella asiatica

The optical density results of the MTT-test in the cultures obtained were about 1.4 times higher in ECA-treated fibroblasts compared to the control culture on day 7, and 1.6 times higher on day 8. If we compare the intensity of formazan formation in fibroblast culture after the addition of EFV with that of control, the values obtained were 2-fold higher on both days 7 and 8. Analysis of MTT-test values in cell cultures treated with plant extracts showed that the results were 1.5 times higher for EFV on day 7, and 1.3 times higher on day 8 compared to the results for ECA-treated fibroblasts.

DISCUSSION:

The resulting plant extract of Ferula asafoetida has demonstrated pronounced pharmacological properties, antioxidant and antimicrobial activity, and wound healing ability. A review provides clinical trial data on the beneficial effects of Centella asiatica in the treatment of skin and neurological diseases. The main active ingredient of ECA, the pentacyclic triterpene madecassoside exhibits marked antioxidant, anti-inflammatory and anti-apoptotic effects. Therefore, the extract of this plant was chosen for comparison with EFV¹⁸.

Phenolic compounds of plant origin include a number of different substances, such as phenolic acids, flavonoids, quinones, coumarins, lignans, stilbenes, tannins, as well as nitrogen compounds, vitamins and terpenoids. Polyphenol molecules are among the most important groups of substances that act as free radical traps and primary antioxidants in medicinal plants. The main class of phenolic compounds are such widely distributed substances in the plant world as phenolic acids¹⁹.

In the tissues of Ferula asafoetida L., small amounts of phenolic acids such as vanillic and ferulic acids were detected²⁰. The data obtained by Folin-Chekolteu method of not very significant content of total phenols in methanol extract of Ferula asafoetida L. correlate with the low antiradical activity of the extract. Correspondingly, the higher concentration of polyphenols in Centella asiatica extract explains its greater antiradical activity.

Flavonoids have been found to be the most abundant class of phenols in known medicinal herbs. The basic structure of flavonoids is described as a diphenylpropane derivative containing 15 carbon atoms²¹. Two 6-membered A and B rings, each linked to three carbon units that are part of a third ring. This third carbon ring is a heterocyclic oxygen-containing pyrene molecule. The major flavonoids of Ferula asafoetida L., most belonging to the sesquiterpene coumarin series, were identified²². The Ferula asafoetida L. extract under study showed a fairly high content of total flavonoids by a complexation reaction with aluminium chloride. Numerically, the result was close to the concentration of total flavonoids in the extract of Centella asiatica, whose preparations have been successfully tested clinically.

In plant cells, flavonoids have many different functions. Their role in protecting plants against UV radiation has been emphasized²¹. Separately, sesquiterpene coumarins have been studied for their involvement in plant immunity²³. Significant antimicrobial activity of these substances has been documented, including the inhibition of growth of standard Pseudomonas aeruginosa and Staphylococcus aureus strains by Ferula asafoetida L. extract in agar. Similar activity was exhibited by the comparison extract²⁴. However, when determining the minimum inhibitory concentration (MIC) by microdilution in broth, the MIC of Centella asiatica extract against Pseudomonas and the MIC of Ferula asafoetida L. extract for both strains were measurable, whereas the MIC of ECA against Staphylococcus aureus exceeded the tested range of 0–1000 µg/ml. These findings correlate with previous studies, including the high antimicrobial activity of Ferula asafoetida extract against Staphylococcus aureus reported elsewhere²⁵.

The observed patterns can be explained by previous findings. The major flavonoid of Ferula asafoetida and coumarin from Centella asiatica were found to inhibit quorum-sensing processes in the P. aeruginosa QSIS2 biosensor strain. This process mediates intercellular communication of the microorganism and regulates the expression of several phenotypes, including biofilm formation, virulence factor production, and motility. In addition, coumarin restored protease and pyocyanin production and inhibited biofilm formation in titrant microplates in various P. aeruginosa strains²⁶. The results confirming the significant antioxidant and antimicrobial activity of Ferula asafoetida L. extract have enabled its use for wound healing. Two models have been developed for possible applications of the extract. In model A, traditional external application in the form of an aseptic dressing with 4% Vaseline ointment was assumed. A fibroblast culture transplantation model was proposed to study the cellular mechanisms of the effects of the active components of Ferula asafoetida L.

In the control group of experiment Series A, no treatment was applied, and the wound healed naturally. The application of dressings with 4% herbal extracts on Vaseline accelerated the healing process by 5 days in all experimental animals. However, there was no significant difference between the application of EFV and ECA in the experimental groups. On the 14th day, epithelialisation and granulation were more pronounced in the ointment dressed rats than in the control group, and the wound was completely healed by the 20th day. Such results suggest that an ointment based on Ferula asafoetida L., extract has at least as much wound-healing properties as an ointment with Centella asiatica.

The findings correlate with a study on wound healing using an ointment containing an extract from a similar plant of the genus Ferula L., namely Ferula persica. The extract was found to have high anti-inflammatory activity by reducing the number of neutrophils and lymphocytes and by inhibiting the regulation of the TNF-α gene and COX-2 protein. In addition, Ferula persica extract has been shown to be able to increase the number of fibroblasts and collagen density in the wound, in addition to increasing the expression of the TGF-β gene²⁷. Based on this results, it was decided to investigate the effects of EFV on fibroblast culture. Fibroblasts, which are the main cellular component of the dermis, regulate skin physiology. During the proliferative phase of wound healing (4-7 days), fibroblasts produce various components of the extracellular matrix, including collagen, and then generate granulation tissue. A review shows that re-epithelialisation occurring in this phase also promotes fibroblast proliferation and migration²⁸.

A statistically significantly higher activity of EFV when added to fibroblast culture compared to ECA was reported from an in vitro MTT test. These results are supported by a study on one of the main components of Ferula asafoetida L. extract, ferulic acid. Ferulic acid encapsulated in nanostructured lipid carriers to increase its bioavailability has demonstrated the ability to stimulate fibroblast proliferation and migration during wound healing. These data are particularly important in the context of the fact that in the comparison preparation, Centella asiatica, extract, ferulic acid is absent and madecassoside is the main biological ingredient²⁹.

In experiment Series B, the presence of a silicone ring prevented the creation of secondary tension, but promoted the physiological effects of transplanted fibroblasts after the addition of plant extracts in vivo. After transplantation of the treated cell cultures onto the wound surface of experimental animals, no significant differences in the rate of wound healing were observed when Ferula asafoetida L. extract was added compared to the preparation of Centella asiatica. However, it should be noted that on day 7 of the wound process, which corresponded to day 4 after fibroblast transplantation, there was a significant difference in the formation of the skin defect. Group 1B animals transplanted with untreated fibroblasts had a more severe skin defect compared to Group 2B (fibroblasts with ECA) and Group 3B (fibroblasts with EFV). The lack of a pronounced advantage in the use of the obtained Ferula asafoetida extract in in vivo experiments compared to the Centella asifoetida extract can be explained primarily by the lower AOS obtained for Ferula asafoetida L. Clearly, the antiradical properties of plant extracts play a leading role in clearing the wound of dead cells and their degradation products. Polyphenols and flavonoids can both act as traps for free radicals and produce limited amounts of them. It has been shown that reactive oxygen species such as hydrogen peroxide, in moderate amounts, trigger the MAP kinase cascade in cells. MAP-kinase signalling pathways are responsible for cell proliferation and apoptosis processes depending on the conditions. In this context, the relatively low content of total phenols determined in EFV by the Folin-Chekolteu method can be seen as a negative factor in the in vivo wound healing processes³⁰.

CONCLUSIONS:

By mixing dried latex and whole Ferula asafoetida plants obtained both in the middle of the growing season and at the end of it, an extract with pronounced pharmacological properties can be obtained. The relatively low content of total phenols in the extract of Ferula asafoetida L. correlates with its not very significant antiradical activity compared to the reference preparation, Centella asiatica extract. A fairly high content of total flavonoids ensures a pronounced antimicrobial capacity of Ferula asafoetida extract against standard strains of P. аeruginosa and S. Аureus, this makes it possible to use the active ingredients of the plant for the treatment of wounds. A comparison of the wound closure processes in the experiment with the application of dressings with 4% Vaseline ointment based on a plant extract revealed no obvious advantages or pronounced disadvantages with Ferula asafoetida L. material in relation to Centella asiatica.

A further justification for the use of the plant studied as a wound-healing agent is the statistically significantly higher viability of the fibroblast cell culture in vitro. According to the MTT-test, the metabolic activity of fibroblasts 48 hours after the addition of Ferula asafoetida L. extract was significantly higher than the MTT-test for cultures treated with Centella asiatica, extract and for control cell cultures. In in vivo experiments, after transplantation of treated fibroblast cultures to the damaged surface, the intensity of wound healing in animals using Ferula asafoetida extract coincided with that of the reference drug. This result can also be considered positive, as Centella asiatica extract has been successfully tested clinically as a wound-healing agent.

The low antioxidant capacity of Ferula asafoetida L. extract does not give it an advantage in its use for in vivo, wound healing, either in the form of dressings with 4% Vaseline ointment or by treating fibroblast cultures with it. The proven antimicrobial activity and positive stimulation of fibroblast culture in vitro give grounds for further development and research of preparations based on the plant extract of Ferula asafoetida for possible use in the treatment of injuries and burns. It can be assumed that the additional enrichment of the studied extract with antioxidant components should enhance its anti-inflammatory and wound-healing effects.

CONFLICT OF INTEREST:

The authors declare no conflict of interest.

ACKNOWLEDGMENTS:

The authors would like to thank the staff of the Department of Pharmacognosy and Pharmaceutical Technology at S.D. Asfendiyarov Kazakh National Medical University for their technical support. We are also grateful to the laboratory team for their assistance with cell culture and antimicrobial testing.

REFERENCES:

1. Tleubayeva MI, Abdullabekova RM, Datkhayev UМ, Ishmuratova MY, Alimzhanova MB, Kozhanova KK, Seitaliyeva AM, Zhakipbekov KS, Iskakova ZB, Serikbayeva EA, Flisyuk EV. Investigation of CO2 Extract of Portulaca oleracea for Antioxidant Activity from Raw Material Cultivated in Kazakhstan. Int J Biomater. 2022 Apr 22; 2022: 6478977. doi: 10.1155/2022/6478977. PMID: 35497070; PMCID: PMC9054492.

2. Shertaeva C, Tulemissov S, Botabayeva R, Blinova O, Mamytbayeva K, Zhanabayev N, Ibragimov G, Datkhayev U, Zhakipbekov K. Improvement of medicine provision of patients with a chronic obstructive lung illness on the basis of pharmaceutical and economical investigations. Life Sci J. 2014; 11(9s): 16–23.

3. Datkhayev U, Shopabaeva A, Zhakipbekov K, Shertaeva C, Umurzakhova G, Sultanbekov A, et al. Determination of seasonal demand for pharmaceutical staff. Int J Pharm Sci Rev Res. 2016; 36(2): 105–111. Available at: https://globalresearchonline.net/journalcontents/v36-2/18.pdf

4. Umurzakhova G, Sultanbekov A, Issatayeva N, Zhakipbekov K, Shopabaeva A, Shertaeva C, Datkhayev U. Communication skills as one of the main competences of pharmacists. Ann Trop Med Public Health. 2018; 11(3): 62. Available at: https://link.gale.com/apps/doc/A608296165/AONE

5. Turgumbayeva A, Zhanat T, Zhakipbekov K, Kalykova A, Kartbayeva E, Mombekov S, et al. A review on the medicinal plant Echinops ritro species: phytochemistry and biological activity. Farmacia. 2023; 71(3): 455–462. doi: 10.31925/farmacia.2023.3.2

6. Turgumbayeva A, Zhakipbekov K, Shimirova Z, Akhelova S, Amirkhanova A, Koilybayeva M, Seitimova G, Abdambayev D. Study of phytochemical compounds of Plantago major leaves grown in Kazakhstan. Pharmacia. 2022; 69(4): 1019–1026. doi: 10.3897/pharmacia.69.e96526

7. Rakhymbayev N, Datkhayev U, Sagindykova B, Myrzakozha D, Zhakipbekov K, Iskakbayeva Z. Component composition and antimicrobial activity of subcritical CO₂ extract of Ferula asafoetida L., growing in the territory of Kazakhstan. ScienceRise: Pharm Sci. 2023; 2(42): 82–91. doi: 10.15587/2519-4852.2023.266654

8. Mombekov S, Rakhymbayev N, Zhakipbekov K, Sagindykova B, Akhatova S, Amirov M, et al. Study of the chemical components of CO₂ extracts from the underground part of Ferula Asafoetida L. ScienceRise: Pharm Sci. 2025; 1(53): 115–122. doi: 10.15587/2519-4852.2025.323987

9. Rakhimov S. Biological and morphological characteristics of Ferula foetidissima Regel et Schmalh. and its cultivation in Tajikistan. Dushanbe: Donish; 2009.

10. Imanbayeva AA, Sarsenbayev KN, Sagyndykova M. Anatomic structure of above and underground organs of Ferula foetida (Bunge) Regel in Mangistau natural populations. Siberian Ecol J. 2015; 22(6): 899–908.

11. Kumar A, Kumar R, Singh S, Sharma S, Kumar S. Evaluation of Ferula assa-foetida accessions for germination parameters under cold stratification to overcome seed dormancy and effect of media mixtures on seedling growth. Preprint, Research Square; 2020. doi: 10.21203/rs.3.rs-112608/v1

12. Sagyndykova MS, Imanbayeva AA, Ishmuratova MY. Study of population structure of Ferula foetida (Bunge) Regel in the conditions of Mangystau region. Bull Karaganda Univ. 2019; 2(94): 159–169.

13. Amalraj A, Gopi S. Biological activities and medicinal properties of asafoetida: A review. J Tradit Complement Med. 2017; 7(3): 347–359.

14. Li D, Rui YX, Guo SD, Luan F, Liu R, Zeng N. Ferulic acid: A review of its pharmacology, pharmacokinetics, and derivatives. Life Sci. 2021; 284: 119921.

15. Appendino G, Mercalli E, Fuzzati N, Arnoldi L, Stavri M, Gibbons S, Ballero M, Maxia A. Antimycobacterial coumarins from the Sardinian giant fennel (Ferula communis). J Nat Prod. 2004; 67(12): 2108–2110.

16. Fialova SB, Rendekova K, Mucaji P, Nagy M, Slobodnikova L. Antibacterial activity of medicinal plants and their constituents in the context of skin and wound infections, considering European legislation and folk medicine – A review. Int J Mol Sci. 2021; 22(19): 10746.

17. Kancherla N, Anusha D, Chitra K, Ravi Babu K. Quantitative estimation of total phenols, flavonoids and antioxidant potential of Cayratia auriculata (in-vitro study). Res J Pharm Technol. 2022; 15(10): 4570–4576. doi: 10.52711/0974-360X.2022.00767

18. Sun B, Wu L, Wu Y, Zhang C, Qin L, Hayashi M, Kudo M, Gao M, Liu T. Therapeutic potential of Centella asiatica and its triterpenes: A review. Front Pharmacol. 2020; 11: 568032. doi: 10.3389/fphar.2020.568032

19. Purushotham K, Nandeeshwar P, Srikanth I, Ramanjaneyulu K, Himabindhu J. Phytochemical screening and in-vitro antioxidant activity of Senna occidentalis. Res J Pharm Technol. 2019; 12(2): 549–552. doi: 10.5958/0974-360X.2019.00097.0

20. Iranshahy M, Iranshahi M. Traditional uses, phytochemistry and pharmacology of asafoetida (Ferula assa-foetida oleo-gum-resin) – A review. J Ethnopharmacol. 2011; 134(1): 1–10.

21. Roy A, Khan A, Ahmad I, Alghamdi S, Rajab BS, Babalghith AO, Alshahrani MY, Islam S, Islam MR. Flavonoids: A bioactive compound from medicinal plants and its therapeutic applications. Biomed Res Int. 2022; 2022: 5445291.

22. Abd El-Razek MH, Ohta S, Ahmed AA, Hirata T. Sesquiterpene coumarins from the roots of Ferula assa-foetida. Phytochemistry. 2001; 58(8): 1289–1295.

23. Gao J, Li T, Jiao L, Jiang C, Chen S, Huang L, Liu J. Metabolome and transcriptome analyses identify the plant immunity systems that facilitate sesquiterpene and lignan biosynthesis in Syringa pinnatifolia Hemsl. BMC Plant Biol. 2022; 22: 132.

24. Kavoosi G, Rowshan V. Chemical composition, antioxidant and antimicrobial activities of essential oil obtained from Ferula assa-foetida oleo-gum-resin: Effect of collection time. Food Chem. 2013; 138(4): 2180–2187.

25. Niazmand R, Razavizadeh BM. Ferula asafoetida: Chemical composition, thermal behavior, antioxidant and antimicrobial activities of leaf and gum hydroalcoholic extracts. J Food Sci Technol. 2021; 58(6): 2148–2159.

26. Zhang Y, Sass A, Van Acker H, Wille J, Verhasselt B, Van Nieuwerburgh F, Kaever V, Crabbe A, Coenye T. Coumarin reduces virulence and biofilm formation in Pseudomonas aeruginosa by affecting quorum sensing, type III secretion and C-di-GMP levels. Front Microbiol. 2018; 9: 1952. doi: 10.3389/fmicb.2018.01952

27. Huang L, Wang M, Ebrahimzadeh MA, Jafari A, Jiang K. Stereological and molecular studies on the effects of Ferula persica extract on wound healing in rats. Vet Med Sci. 2022; 8(1): 318–327.

28. Masson-Meyers DS, Andrade TAM, Caetano GF, Guimaraes FR, Leite MN, Leite SN, Frade MAC. Experimental models and methods for cutaneous wound healing assessment. Int J Exp Pathol. 2020; 101(1–2): 21–37.

29. Carbone C, Caddeo C, Grimaudo MA, Manno DE, Serra A, Musumeci T. Ferulic acid-NLC with Lavandula essential oil: A possible strategy for wound-healing? Nanomaterials. 2020; 10(5): 898.

30. Son Y, Kim S, Chung HT, Pae HO. Reactive oxygen species in the activation of MAP kinases. Methods Enzymol. 2013; 528: 27–48.

Received on 14.07.2025 Revised on 08.11.2025

Accepted on 06.01.2026 Published on 01.07.2026

Available online from July 04, 2026

Research J. Pharmacy and Technology. 2026;19(7):3219-3226.

DOI: 10.52711/0974-360X.2026.00458

This work is licensed under a Creative Commons Attribution-NonCommercial-ShareAlike 4.0 International License. Creative Commons License.

Experiment		Series A			Series B
Animal group		1 (n=7)	2 (n=7)	3 (n=7)	1 (n=6)	2 (n=6)	3 (n=6)
Day 0	S of wound, mm	324±48	324±36	320±53	315±45	310±36	320±38
Day 0	% of wound closure	0	0	0	0	0	0
Day 7	S of wound, mm	324±33	33±8	33±7	30±7	66±7*	66±7*
Day 7	% of wound closure	0	10±2	10±2	9,5±2	21±2	21±2
Day 14	S of wound, mm	160±10	204±8	206±7	200±11	248±10*	254±10*
Day 14	% of wound closure	50±3	63±2.5	64±2	64±3.5	80±3	79±3
Day 21	S of wound, mm	286±7	0	0	0	0	0
Day 21	% of wound closure	88±2	100	100	100	100	100