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Identification of the Anti-inflammatory and Wound Healing Effects of Cordyceps militaris Extract in Animal Models

Ouafa Rebai*, Nesrine Ouda Amari, Fatiha Missoun

Department of Biology, University Abdelhamid Ibn Badis, Mostaganem, Algeria

Abstract: Cordyceps militaris is one of the most well known medicinal entomopathogenic fungi and as such has been widely used for the treatment of various diseases. However, the pharmacological and biochemical activities of C. militaris have not been clearly elucidated. The evaluation of the anti-inflammatory activity of C. militaris was performed by the test for plantar edema induced by injection of carrageenan in mice. The percentage inhibition of edema and the percentage increase in paw edema volume were then evaluated. Wound healing effect of C. militaris extract was studied on an excision wound model in rats. Changes in wound area were measured regularly and the rate of wound contraction calculated, then the histopathological recovery was evaluated. The results in all animals were not shown signs of toxic eﬀects, moribund, and mortality. Oral administration of C. militaris extract reduced significantly after 2 hours the paw edema induced by carrageenan in mice (8.96±2.83%, 6.25±2.70% and 4.92±2.22 % at doses of 100, 200 and 300mg/kg BW respectively); the histological study confirmed the decrease in inflammatory response. Topical application of C. militaris ointments at 5% and 10% showed an enhance rate of contraction and significantly decreased wound area than control group. With histological evidence of more collagen formation in the skin and early epithelization period with low scar area. C. militaris mushrooms deﬁnitely deserve to be considered as functional foods and also have great potential for medicinal use.

Keywords: Cordyceps militaris, anti-inflammatory, wound healing, mice, rat

Received July 23, 2023; Revised August 24, 2023; Accepted August 31, 2023

Citation: Ouafa Rebai, Nesrine Ouda Amari, Fatiha Missoun, Identification of the Anti-inflammatory and Wound Healing Effects of Cordyceps militaris Extract in Animal Models. Journal of Food and Nutrition Research, 11 (8). 549-556. January 2023. doi:10.12691/jfnr-11-8-5

1. Introduction

Therapeutic mushrooms continue to be a significant part of human culture and have intriguing health benefits [1]. With about 750 species, the genus Cordyceps is one of the largest genera in the Clavicipitaceae family. It is also one of the most diversified in terms of variation in species, morphology, and ability to adapt to a variety of hosts [2]. The medicinal mushroom Cordyceps militaris found in East Asia, has been widely consumed in China for medication purposes since ancient times (3000 years) [3,4]. Due to its rarity, many investigators have been devoted to focused on the artiﬁcial cultivation of C. militaris using cutting-edge technology [5].

The mycelium and fruiting bodies of C. militaris are ideal food supplements and pharmaceutical agents and conserve as a good source of prohealth substances potentially bioavailable for humans. Studies on the fruiting bodies and mycelia of C. militaris have shown the presence of biologically active substances, such as γ-aminobutyric acid, ergothioneine, sterols (ergosterol), statins (lovastatin), phenolic compounds (including phenolic acids and ﬂavonoids), vitamins, and bioelements [6]. Cordycepin, peptides, polysaccharides, and other active compounds produced from C. militaris will be a key factor in the development of green pharmacognosy and pharmacology [7]. These numerous bioactive compounds obtained from this fungus have been looked as responsible for their biological and therapeutic properties, with their antitumor, and immunomodulatory effects [8,9,10,11,12], anti-inflammatory activity [13], and antioxidant activity [14].

Inflammation plays a crucial role in biological processes that are triggered by a variety of stimuli and harmful elements, including UV radiation, irritants, infections, and cell damage. Redness, a rise in body temperature, pain, and changes in physiological processes at infected locations are the key characteristics of inflammation [15,16]. Antioxidant function, transcription factors, matrix metalloproteinases, complement cascade characteristics, and adhesion molecules including intercellular adhesion molecule-1 (ICAM-1), selectin, and vascular cell adhesion molecule-1 (VCAM-1) are all components of inflammatory responses. Additionally, C. militaris and its contents control the expression of inflammatory genes as well as the actions of proinflammatory enzymes.

Cordyceps species, its extracts, and bioactive constituents have been related with cytokine production such as interleukin (IL)-1β, IL2, IL-6, IL-8, IL-10, IL-12, and tumor necrosis factor (TNF)-α, phagocytosis stimulation of immune cells, nitric oxide production by increasing inducible nitric oxide synthase activity, and stimulation of inﬂammatory response via mitogen-activated protein kinase pathway [17]. Pre-clinical research has revealed several pharmacological effects, including antioxidant, anti-cancer, antihyperlipidemic, antidiabetic, anti-fatigue, anti-aging, hypocholesterolemic, hypotensive, vasorelaxant, anti-depressant, aphrodisiac, and kidney protection. The bioactive substances found in Cordyceps, such as nucleosides, sterols, flavonoids, cyclic peptides, phenolics, bioxanthracenes, polyketides, and alkaloids, are associated with these biological activities [18]. The research involving C. militaris-derived polysaccharides has increased rapidly, particularly in their structural characterization and pharmaceutical activities including antioxidant, immunomodulatory, and antitumor activities [19].

In recent years, naturals are being employed in wound treatment because of their ability to promote blood clotting, fight infection, and accelerate wound healing. These bioactive agents usually modulate one or more phases during healing processus [20]. Extracts from medicinal mushrooms and their metabolites have been verified for wound treating with contribution to different mechanisms of the healing process [21]. Wounds are physical injuries that result in an opening or breaking of the skin. Skin wound healing shows an extraordinary cellular function mechanism, unique in nature and involving the interaction of several cells, growth factors, and cytokines [22]. The wound healing of mushrooms has been also increasingly reported, linked to their richness in polysaccharides and phenolic compounds [23].

Therefore, substantial attention has recently been given to the development of natural compounds for being employed in anti-inﬂammatory and wound treatments with greater safety and minimal side effects. In the current study, research activities related to anti-inflammatory and wound healing exhibited by C.militaris extract was conducted in animal models.

2. Materials and Methods

2.1. Fungal Material

Aqueous extract of the medicinal Mushroom Cordyceps militaris (L.) (Sk gold) was purchased from the Institute of natural products of Hanoi, Vietnam Academy of science and technology. The determination of the concentrations of mineral elements in C. militaris extract was measured by a graphite furnace atomic absorption spectroscopy (AA-7000, SHIMADZU).

2.2. Animal Material

Female mice NMRI (25-30g) and male Wistar rat (250-270g) were obtained from the Pasteur institute (Algiers, Algeria). Animals were kept in polyacrylic cages and maintained under standard housing conditions (room temperature at 25±2°C with 12:12 light: dark cycles) and water ad libitum. Food was provided by dry pellets. This study was approved in accordance with the Algerian Legislation (Law Number 17-457/2019) inherent to the protection of animals designed to experimental and other scientific purposes as well as with the guidelines of the Algerian Association of Experimental Animal Sciences (AASEA).

2.3. Acute Oral Toxicity

Acute toxicity study of C. militaris was assessed in both mice and rats by using an acute oral toxic class method of Organization of Economic Co-operation and Development (OECD), guidelines (OECD/OCDE, 2000). Briefly, the C. militaris powder were calculated at gradual doses (300 mg, 500 mg, 1000mg/kg body weight) for mice and a unique dose (5000 mg/kg body weight) for rats and freshly mixed with distilled water as a vehicle prior to administration. All animals were fasted 18 h before testing and divided into two groups, the control group received only the vehicle, and the test group received C. militaris orally by gavage using a stainless steel stomach tube. Animals were then observed for any manifestation of toxicity, increase in locomotor activity, salivation, convulsions, coma, and death at ﬁrst 15, 30, and 60 minutes then daily for 14 days.

2.4. In vivo Anti-inflammatory Activity

Anti-inflammatory properties of C. militaris were performed according to the recommended methods of Alwashli et al. [24] and Bignotto et al. [25]. A total of thirty healthy female mice NMRI were fasted 18 h before testing and divided into five groups of six mice each. The duration of experimentation was 6 hours, with each group receiving the experimental solutions orally as follows: In group 1 - control group (Physiological water, NaCl 0,9%), group 2 - (Diclofenac dissolved in NaCl 0,9% with concentration of 10ml/kg), group 3 - Low dose of aqueous extract of C. militaris (100mg/Kg body weight of C. militaris dissolved in NaCl 0,9% ), group 4 – middle and group 5 - high dose (200mg/Kg and 300mg/Kg body weight of C. militaris dissolved in NaCl 0,9%, respectively). The dosage of administration to each animal was calculated based on the body weight of the animal prior to administrate at a constant volume not exceed 1 ml per 100 g body weight. One hour after the administration of the treatments, each animal in all groups received, by sub-plantar injection in the right hind paw, 0.1 ml of a 0.1% carrageenan a highmolecular-weight polysaccharide induced paw edema model dissolved in 0.9% NaCl.

The percentage increase in paw edema volume (% AUG) for each group of mice was calculated by measuring the diameter of the paw, using a digital micrometer before and after induction of inflammation at intervals of one hour to six hours, it is given in the equation (1) below. Then the percentage inhibition of edema (% INH) was calculated for each group of mice treated relative to the control as given in the equation (2).

[picture] | (1)

Dn: Diameter of the leg after the injection of carrageenan and DO: Diameter of the leg before the injection of carrageenan.

[picture] | (2)

2.5. In vivo Wound Healing Activity

The preparation of the ointments to be applied was carried out by drawing up first the cream, wich was prepared by mixing an aqueous phase (xanthan gum in distilled water) with a fatty phase (sunflower oil, emulsifying wax, and conservative). The base cream was then added to the medicinal mushroom extract under sterile conditions to create the C. militaris ointments with 5 and 10% densities. The C. militaris ointments were topically applied to assess any manifestation of inflammation (erythema and edema).

Rats were anesthetized with an intraperitoneal (i.p.) injection of ketamine (50 mg/kg) and diazepam (1 mg/kg), and after the back was shaved, an area of about 2 cm² was marked by a standard ring above the spin. Full-thickness of the marked skin was then cut carefully. 24 hours after creation of open excision wounds, the animals were divided into five groups of five rats each. The rats were subjected to daily application of the experimental preparations in to the wound at the same time once a day for 21 days as follows: In group 1 - control group (non treated rats), group 2 – placebo (rats treated with 0,5 g of base cream), group 3 – standard (rats treated with 0,5 g of madecassol), group 4 – low dose (rats treated with 0,5 g of C. militaris ointment 5%), group 5 - high dose (rats treated with 0,5 g of C. militaris ointment 10%).

The evolution of the lesions was evaluated using the healing assessment parameters which are epithelization, exudate, erythema and scar. The wounds were observed and photographed every 3 days at the same time and under the same conditions until their complete closure. To determine the wound area, a transparent acetate paper was placed directly on the wound, and its outline was traced with a fine-tipped marker and the number of squares in the outline were counted [26]. Changes in wound area were monitored planimetrically by tracing the wound margin on graph paper regularly and the rate of wound contraction was calculated as given in the equation (3).

[picture] | (3)

*Healed area = original wound area − present wound area.

2.6. Histological Study

Six hours after the induction of inflammation in mice and after the 21st day for histopathological examination of skin in rats, the animals were euthanized using CO₂ chamber and the animals death was confirmed by assessing the heart rate and respiration. The process of euthanasia was conducted following the AVMA guidelines for the euthanasia of animals: 2020 edition. Tissue samples were taken, fixed in 10% buffered formalin, processed, decalcified with 2% nitric acid solution for three hours, blocked with paraffin, then sectioned into 5 μm sections, and stained with hematoxylin and eosin.

2.7. Statistical Analysis

Statistical comparison was performed using one-way analysis of variance (ANOVA) followed by the paired Student’s t-test in the XLSTAT software, and difference was considered significant for P<0.05.

3. Results

3.1. Anti-inflammatory Activity

The evaluation of the anti-inflammatory activity of C. militaris was performed by the test for plantar edema induced by injection of carrageenan in mice. The percentage increase in paw edema volume (%AUG) that carrageenan administration significantly increased was calculated for all treated groups of mice (Figure 1). Oral administration of the aqueous extract of C. militaris at doses of 100, 200, and 300mg/kg BW seems to significantly reduce carrageenan-induced edema at the second hour (8.96±2.83%, 6.25±2.70% and 4.92±2.22 % respectively). At the third hour, this decrease is greater in mice treated with high doses (3.13±1.80% at 200mg/kg BW and 1.27±0.9% at 300mg/kg BW), values which remained significantly reduced compared to the values of the second, third and fourth hour in the group treated at the highest dose of 300mg/kg BW (P < 0.01).

The results of the percentage inhibition of edema (% INH) indicate that after the first and second hour (Figure 2), the treatment with the oral administration of the C. militaris extract at the doses 200 and 300 mg/kg BW caused a significant inhibitory activity in carrageenan induced paw inflammation compared to the group treated with the extract at a dose of 100 mg/kg BW and standard group (P <0.01).

Figure 1. The percentage increase in paw edema volume (%AUG). Mice were divided randomly into five groups: Control Carrageenan group, Diclofenac group (50mg/kg), C. militaris (D1) groups (100mg/kg), C. militaris (D2) groups (200mg/kg) and C. militaris (D3) groups (300mg/kg). Results are expressed as the mean±SD (n=6 animals per group). *p<0.05, **p<0.01, ***p<0.001 were considered significant when compared with the Control carragenan group.

Figure 2. Effect of C. militaris extract on percentage inhibition of edema (% INH). Mice were divided randomly into five groups : Control carrageenan group, Diclofenac group (Mice were divided randomly into five groups: Control Carrageenan group, Diclofenac group (50mg/kg), C. militaris (D1) groups(100mg/kg), C. militaris (D2) groups (200mg/kg) and C. militaris (D3) groups (300mg/kg). Results are expressed as the mean±SD (n=6 animals per group). *p<0.05, **p<0.01, ***p<0.001 were considered significant when compared with the standard group (Diclofenac)

3.2. Wound Healing activity

Irritation test of C. militaris extract observed after application on the backs of rats indicated no signs of inflammation (Erythema or edema). The primary cutaneous irritation index obtained allows this extract to be considered as non-irritating (data not shown).

The Wound contraction assessed by comparison of the score’s means of macroscopic appearance of the wounds from day 0 to day 21 revealed an improvement in the appearance of the wounds with a progressive reduction in erythema and exudate observed in all groups (Table 1). Regarding erythema, a significant appearance was shown from day 12 (P<0.05) and day 3 (P<0.001) for the placebo and Madecassol groups, respectively, which then tended to decrease beyond day 15 until day 21. Topical application of C. militaris ointment (5%) showed a significant appearance of erythema compared to the control group from day 6, significance was observed (P<0.01) until day 21. For the group of C. militaris (10%), this appearance became significant (P<0.01) from day 9 to day 21. The exudate present in the control and placebo groups in the first 3 days was absent in each of the Madecassol and C. militaris (5% and 10%) groups. The appearance of buds began on day 3 in all groups and increased in each of the control and placebo groups until day 9, for the Madecassol and C. militaris (5% and 10%) groups the increase noticed until day 12 was higher than that of the control and placebo. The epithelization phase began on day 3 in all groups, it increased in each of the control and placebo until day 9, then gradually decreased in the following days. This increase was higher in the Madecassol and C. militaris groups (5% and 10%), from day 12 a rapid gradual decrease was noticed until the last day of observation. Scab formation started from day 3 in all treated rats, then increased in each of the control and placebo lots until day 9. In the treated groups of madecassol and C. militaris (5% and 10%) a higher increase was noticed (P<0.01) until day 12, followed by a more rapid gradual decrease observed until the last day of the test.

Table 1. Effect of topical aplication of ointments on erythema, exudate, appearance of buds, epithelization period and scar area in excision wound. *p<0.05, **p<0.01, ***p<0.001 were considered significant when compared with the Control group

Macroscopic aspect of wounds appeared less inflamed with the evolution of the days, those treated with C. militaris showed the best evolution compared to the other treatments (Figure 3). Rates of wound contraction observed at day 21 were 78,14%, 82,07%, 97,28%, 97,86%, and 98,68% in control rats, placebo, Madecassol, C. militaris 5% and C. militaris 10% respectively (Figure 4). The wounds treated with Madecassol and C. militaris (5%) ointments showed a significant surface area compared to the control in the first 3 days (P<0.05), from day 6, a significant increase in surface compared to the control (P<0.001). From day 9 to day 12 a significant increase in surface area was shown (P<0.01), after day 15 until day 21 a highly significant increase (P<0.001) was observed. Wounds treated with C. militaris ointment (10%) revealed a highly significant increase in surface area within 21 days compared to the control (P<0.001).

Histology of excision biopsy of skin wound showed healed skin structures in both madecassol and C. militaris (5% and 10%) treated groups, in which were observed after day 21 of the topical application of the ointment treatment, the dermis and epidermis with formation of the amount of ground substance in the granulation tissue (Figure 5).

Figure 3. Photographic representation of contraction rate showing percent wound contraction area on different post excision days of control, placebo, madecassol, C. militaris 5% and 10% treated rats.

Figure 4. Rates of wound contraction. Results are expressed as the mean±SD (n=5 animals per group). *p<0.05, **p<0.01, ***p<0.001 were considered significant when compared with the Control group.

4. Discussion

The detection of mineral elements using atomic absorption method showed that the content of manganese (Mg), cadmium (Cd), copper (Cu), and zinc (Zn) in C. militaris extract, respectively, is 2.0227 μg/g, 17.9078 μg/g, 0.0187 μg/g and 0.1346 μg/g. The content of heavy metals in cultivated fruiting bodies of C. militaris is within the limits of Chinese national food safety standard [27]. Moreover, trace elements are essential to immune regulation and infection prevention [28].

The acute oral toxicity of C. militaris in rats (5000 mg/kg BW) and mice (300 mg, 500 mg, 1000mg/kg BW) was not shown signs of toxic, moribund and mortality in all animals. Thus, the health observation, feed and drinking water consumptions, was normal. These findings are consistent with the study by Long et al. [29], who worked on the toxicity of Chinese Cordyceps. C. militaris was recently classiﬁed in GHS category 5 or unclassiﬁed, the LD50 cut oﬀ at 5000 mg/kg body weight to inﬁnity (∞) [30]. In our study, the analyzed parameters of the hematological analysis and clinical biochemistry analysis were not shown toxic to C. militaris in Wistar rats (data not shown).

In the first part of this study, the anti-inflammatory activity of C. militaris was evaluated in mice at dose level 100, 200, and 300 mg/kg BW. Results obtained showed that the aqueous extracts of C. militaris appreciably reduce the induced paw edema by carrageenan 1%, this inhibition is comparable to that of Diclofenac. Carrageenan is a mucopolysaccharide that induces local inflammation following its injection, the cause of this reaction is tissue damage which induces the synthesis of histamine, prostaglandins, leukotrienes, PAF (platelet activating factor), cytokines, NO (monoxide nitrogen) and TNF [31]. In addition, many studies seem to indicate that LPS is a potent macrophage activator related to the release of a variety of cytokines produced by cells and trigger or enhance the specific inflammation response [32].

Figure 5. Histopathology of skin ×40. A: Control rat. B: Base cream treated rat (Placebo). C: Madecassol treated rar. D : C. militaris (5%) treated rats. E: C. militaris (10%) treated rats. LC: Langerhas Cells; KC: Keratinocytes; MEL: Melanocytes; COLL: Collagene.

Microscopic observation of the treated mice with the aqueous extract of C. militaris at the lowest dose (100mg/kg BW) and the highest dose (200mg/kg BW) showed a skin covering within the normal histological limit with almost total disappearance of the inflammatory infiltrate, at the highest dose (300mg/kg BW) the disappearance of edema and tissue congestion with a decrease in the intensity of the inflammatory infiltrate were observed (data not shown). The richness of the aqueous extract of C. militaris in different chemical constituents can justify this anti-inflammatory activity [33]. The actual mechanisms involved in anti-inﬂammatory responses include antioxidant activity, transcription factors, matrix metalloproteinases, complement cascade properties, and adhesion molecules such as intercellular adhesion molecule-1 (ICAM-1), selectin, and vascular cell adhesion molecule-1 (VCAM-1) [7]. In subsequent in vitro tests, the anti-inﬂammatory activity of C. militaris was conﬁrmed to result from the inhibition of the production of proinﬂammatory mediators (NO,TNF-α and IL-6), which were induced by LPS in murine macrophages, the group of bioactive compounds of C. militaris was not deﬁned in the study [34]. Recent studies showed that COX-2 and iNOS gene expressions were suppressed by cordycepin, one of the major components of C. militaris [35].

In the second part, the healing activity of C. militaris was evaluated in Wistar rats. Topical application of the ointment based on aqueous extract of C. militaris did not cause any skin irritation. The results of wound evolution indicate the presence of erythema and exudate in all animals from day 0, which thereafter gradually decreases over 21 days. The application of the ointment based on C. militaris inhibited erythema and exudates, and prevented wound odors, this reduction confirms that this mushroom could act on the inflammatory phase, as demonstrated by Chiu et al. [36] with the implication of the mycelium of C. militaris. As a result of this early anti-inflammatory effect of the ointment is the effective shortening of the re-epithelialization time [37]. In addition, the increase in wound contraction speed for 21 days seems to confirm the effects of the extract. The planimetric study therefore allows a direct quantitative evaluation by calculating the surface of the wound, its evolution over time, and by deducing an assessment of the quality of the granulation tissue. The evaluation of the macroscopic aspect of the wounds, which appear less inflamed with the evolution of the days, suggests that the fungi extract is able to modulate the inflammatory response. Indeed, the percentage of inhibition seems to show that the wounds treated with C. militaris have a better evolution than those treated with Madecassol, placebo and controls. The work of Bahramikia and Yazdanparast [38] also reported that the acceleration of the scarring process may be linked to the anti-inflammatory power by the formation of complexes neutralizing many irritants. The results of the histological study of skin biopsies of rats treated with different ointments confirm the healing effect of the ointment based on C. militaris. The topical application of the ointments with Madecassol and C. militaris (5% and 10%) induced the reconstitution of the organization of the structure of the skin and superficial scarring in the 3 groups of rats, C. militaris at 10% presented the best result. The moderate increase in skin tensile strength in animals after application of the ointment based on C. militaris may be due to the richness of collagen organized into bands.

5. Conclusion

This study evaluated the anti-inflammatory and wound healing potentials of the therapeutic mushroom C.militaris extract in vivo. The findings make C. militaris a suitable functional food for inhibiting inﬂammatory responses such as the regulation of proinﬂammatory cytokines. Moreover, this fungi may be employed in wound treatment because of its ability to accelerate wound healing. The results suggested that C. militaris extract has the potential to be used as topical treatment of skin injuries.

Acknowledgements

The authors are grateful to Abdelhamid Ibn Badis University of Mostaganem for providing facilities and support for the study. This research was funded by the Abdelhamid Ibn Badis University of Mostaganem, and Fundamental Fund (PRFU: D01N01UN270120190001) from the ministry of higher education and scientific research (Algeria).

References

[1]   | Shweta, S., Komal A., Kumar, A, A brief review on the medicinal uses of Cordyceps militaris, Pharmacological Research Modern Chinese Medicine, (7). 100228. 2023.
[2]   | Sung, G.H, Shrestha, B., Han, S.K. and Sung, J.M, Cultural Characteristics of Ophiocordyceps heteropoda Collected from Korea, Mycobiology, 39. 1–6. 2011.
[3]   | Wang, Y., Stata, M., Wang, W., Stajich, J.E., White, M.M. and Moncalvo, J.M, Comparative genomics reveals the core gene toolbox for the fungusinsect symbiosis, MBio, 9( 3). 00636–18. 2018.
[4]   | Xiang, R.K., Zhen, Y., Xiao, J.Z. and Yong, M.Z, Effects of cordyceps polysaccharides on pasting properties and in vitro starch digestibility of wheat starch, Food Hydrocolloids, 102. 1–8. 2020.
[5]   | Cui, J.D, Biotechnological production and applications of Cordyceps militaris, a valued traditional Chinese medicine, Critical Reviews in Biotechnology, 5(4). 475-484. 2015.
[6]   | Chan, J.S., Barseghyan, G.S., Asatiani, M.D. and Wasser, S.P, Chemical composition and medicinal value of fruiting bodies and submerged cultured mycelia of caterpillar medicinal fungus Cordyceps militaris CBS-132098 (Ascomycetes), International Journal of Medicinal Mushrooms, 17. 649–659. 2015.
[7]   | Phull, A., Ahmed, M. and Park, H.J, Cordyceps militaris as a Bio Functional Food Source: Pharmacological Potential, Anti-Inﬂammatory Actions and Related Molecular Mechanisms, Microorganisms, 10. 405. 2022.
[8]   | Shen, H.S., Shao, S., Chen, J.C. and Zhou, T, Antimicrobials from mushrooms for assuring food safety, Comprehensive Reviews in Food Science and Food Safety, 16(2). 316–329. 2017.
[9]   | Meng, X., Li, Y., Li, S., Gan, R.Y. and Li, H.B, Natural products for prevention and treatment of chemical-induced liver injuries, Comprehensive Reviews in Food Science and Food Safety, 17(2). 472–495. 2018.
[10]   | Li, P., Qin, X., Yang, H., Wang, Z.Y. and Lu, D, Therapeutic potentialand biological applications of Cordycepin and metabolic mechanisms in Cordycepin-producing fungi, Molecules, 24(12). 2231. 2019.
[11]   | Guo, Z., Chen, W., Dai, G. and Huang, Y. Cordycepin suppresses the migration and invasion of human liver cancer cells by downregulating the expression of CXCR4, International Journal of Molecular Medicine, 45(1).141–150. 2020.
[12]   | Liu, Y., Guo, Z.J. and Zhou, X.W. Chinese Cordyceps: Bioactive Components, Antitumor Effects and Underlying Mechanism-A Review, Molecules, 27(19). 6576. 2022
[13]   | Jo, L.W., Choi, Y.J., Kim, H.J., Lee, J.Y., Nam, B.H., Lee, J.D, et al., The anti-inflammatory effects of water extract from Cordyceps militaris in murine macrophage, Mycobiology, 38(1). 46–51. 2010.
[14]   | Rao, Y.K., Fang, S.H., Wu, W.S. and Tzeng, Y.M, Constituents isolated from Cordyceps militaris suppress enhanced inﬂammatory mediator’s production and human cancer cell proliferation, Journal of Ethnopharmacology, 131. 363–367. 2010.
[15]   | Chen, X., Wu, G. and Huang, Z, Structural analysis and antioxidant activities of polysaccharides from cultured Cordyceps militaris, International Journal of Biological Macromolecules, 58. 18–22. 2013.
[16]   | Phull, A.R. and Kim, S.J, Fucoidan as bio-functional molecule: Insights into the anti-inﬂammatory potential and associated molecular mechanisms, Journal of Functional Foods, 38. 415–426. 2017.
[17]   | Segal, J.P., Tresidder, K.A., Bhatt, C., Gilron, I. and Ghasemlou, N.J, Circadian control of pain and neuroinﬂammation, Journal of Neuroscience Research, 96.1002–1020. 2018.
[18]   | Jedrejko, K.J., Lazur, J. and Muszynska, B, Cordyceps militaris: An Overview of Its Chemical Constituents in Relation to Biological Activity, Foods, 10. 2634. 2021.
[19]   | Das, G., Shin, H.S., Leyva-Gómez, G., Del Prado-Audelo, M.L., Cortes, H., Singh, Y.D., et al. Cordyceps spp.: A Review on Its Immune-Stimulatory and Other Biological Potentials, Frontiers in Pharmacology, 11. 2020.
[20]   | Miao, M., Yu, W.Q., Li, Y., Sun, Y.L. and Guo, S.D, Structural Elucidation and Activities of Cordyceps militaris-Derived Polysaccharides: A Review, Frontiers in Nutrition, 31(9). 898674. 2022.
[21]   | da Silva, S.M.M., Costa, C.R.R., Gelfuso, G.M., Guerra, E.N.S, Nóbrega, Y.K.M., Gomes, S.M, et al., Wound Healing Effect of Essential Oil Extracted from Eugenia dysenterica DC (Myrtaceae) Leaves, Molecules, 24(2). 2019.
[22]   | Sharifi-Rad, J., Butnariu, M., Ezzat, S.M., Adetunji, C.O., Imran, M., Sobhani, S.R;, et al., Mushrooms-Rich Preparations on Wound Healing: From Nutritional to Medicinal Attributes, Frontiers in Pharmacology, 11. 567518. 2020.
[23]   | Tottoli, E.M., Dorati, R., Genta, I., Chiesa, E., Pisani, S. and Conti, B, Skin Wound Healing Process and New Emerging Technologies for Skin Wound Care and Regeneration, Pharmaceutics, 12. 735. 2020.
[24]   | Alwashli, M., Al Sobarry, Y., Cherrah and Alaoui, K, Anti-inflammatory and analgesic effects of ethanol extract of Dracaena cinnabari balf, as endemic plant in Yemen, International Journal of pharma and biosciences, 3. 96-106. 2012.
[25]   | Bignotto, L., Rocha, J., Sepodes, B., Eduardo-Figueira, M., Pinto, R., Chaud, M., et al., Anti-inflammatory effect of lycopene on carrageenan-induced paw oedema and hepatic ischaemia-reperfusion in the rat, British Journal of Nutrition, 102(1). 126-33. 2009.
[26]   | Balazs, L., Okolicany, J., Ferrebee, M., Tolley, B. and Tigyi, G, Topical application of the phospholipid growth factor lysophosphatidic acid promotes wound healing in vivo, American Journal of Physiology-Regulatory Integrative and Comparative Physiology, 280(2). 466-72. 2001.
[27]   | Fenglin, L., Li, L., Xiaokun,. J, Yan, L. and Hengwei, L, Analysis of seven mineral elements in cultivated fruiting bodies of Cordyceps militaris, E3S Web of Conferences, 185:04019ICEEB. 2020.
[28]   | Li, J., Shen, B., Nie, S., Duan, Z. and Chen, K, A combination of selenium and polysaccharides: promising therapeutic potential, Carbohydrate Polymers, 206. 163–173. 2019.
[29]   | Long, H., Qiu, X., Cao, L., Liu, G., Rao, Z. and Han, R, Toxicological safety evaluation of the cultivated Chinese cordyceps, Journal of Ethnopharmacology, 268. 113600. 2021.
[30]   | Suwannasaroj, K., Srimangkornkaew, P., Yottharat, P. and Sirimontaporn, A, The Acute and Sub-Chronic Oral Toxicity Testing of Cordyceps militaris in Wistar Rats, นิพนธตนฉบับ ว กรมวิทย พ, 63(3). 628-647. 2021.
[31]   | De Garde Elion Itou, R., Sanogo, R., Ossibi, A.W.E., Ntandou, F.G.N., Ondelé, R., Pénemé, B.M., et al., Anti-inflammatory and analgesic effects of aqueous extract of stem bark of Ceiba pentandra Gaertn, Pharmacology and Pharmacy, 5(12). 2014-512121. 2014.
[32]   | Jeong, J.W., Jin, C.Y., Kim, G.Y., Lee, J.D., Park, C., Kim, G.D., et al., Anti-Inﬂammatory Effects of Cordycepin via Suppression of Inﬂammatory Mediators in BV2 Microglial Cells, International Immunopharmacology, 10. 1580–1586. 2010.
[33]   | Ye, H., Wang, Y., Jenson, A.B. and Yan, J, Identification of inflammatory factor TNFα inhibitor from medicinal herbs, Experimental and Molecular Pathology, 100(2). 307-311. 2016.
[34]   | Smiderle, F.R., Baggio, C.H., Borato, D.G., Santana-Filho, A.P., Sassaki, G.L., Iacomini, M., et al., Anti-Inflammatory Properties of the Medicinal Mushroom Cordyceps militaris Might Be Related to Its Linear (1→3)-β-D-Glucan, Plos One, 9(10):e110266. 2014.
[35]   | Jo, W.S., Choi, Y.J., Mm, H.J., Lee, J.Y., Nam, B.H., Lee, J.D., et al., The Anti-Inﬂammatory Effects of Water Extract from Cordyceps militaris in Murine Macrophage, Mycobiology, 38. 46–51. 2010.
[36]   | Chiu, C.P., Liu, S.C., Tang, C.H., Chan, Y., El-Shazly, M., Lee, C.L., et al., Anti-inflammatory Cerebrosides from Cultivated Cordyceps militaris, Journal of Agricultural and Food Chemistry, 64(7). 1540-8. 2016.
[37]   | Ernest, D., Yimta, F., Stéphanie, M.K. and Théophile, D, Activité cicatrisante d’une pomade à base des feuilles de Kalanchoe crenata, International Journal of Research and Analytical Reviews, 6(4). 2349-5138. 2019.
[38]   | Bahramikia, S. and Yazdanparast, R, Phytochemistry and medicinal properties of Teucrium polium L. (Lamiaceae), Phytotherapy Research, 26(11). 1581-93. 2012.
[39]   | Lin, W.H., Tsai, M.T., Chen, Y.S., Hou, R.C.W., Hung, H.F., Li, C.H., et al., Improvement of sperm production in subfertile boars by Cordyceps militaris supplement, American Journal of Chinese Medicin, 35(4). 631-41. 2007.
[40]   | Pooja, P. and Anand, S, Studies on the biology of Cordyceps militaris: A medicinal mushroom from North West Himalaya, KAVAKA, 43. 35-40. 2014.
[41]   | Misset, B. and Desport, J.C, Nutrition et cicatrisation, Actualités Pharmaceutiques, 59. 20-22. 2020.
[42]   | Fontaine, J. and Raynaud-Simon, A, Pressure sores in geriatric medicine: the role of nutrition, La Presse Médicale, 37(7-8). 1150-7. 2008.
[43]   | Ellinger, S. and Stehle, P, Efficacy of vitamin supplementation in situations with wound healing disorders: results from clinical intervention studies, Current Opinion in Clinical Nutrition and Metabolic Care, 12(6). 588-95. 2009.
[44]   | Wang, X., Liu, B., Xu, Q., Sun, H., Shi, M., Wang, D., et al., Feng GHK-Cu-liposomes accelerate scald wound healing in mice by promoting cell proliferation and angiogenesis, Journal of Tissue Repair and Regeneration, 25(2). 270-278. 2017.
[45]   | Jahromi, M.A.M., Zangabad, P.S., Basri, S.M.M., Zangabad, K.S., Ghamarypour, A., Aref, A.R., et al. Nanomedicine and advanced technologies for burns: Preventing infection and facilitating wound healing, Advanced Drug Delivery Reviews, 123. 33-64. 2018.
[46]   | Chow, O. and Barbul, A, Immunonutrition: Role in Wound Healing and Tissue Regeneration, Advanced wound care (New Rochelle), 3(1). 46-53. 2014
[47]   | Borkow, G., Okon-Levy, N. and Copper, J.G, Oxide impregnated wound dressing: biocidal and safety studies, Wounds, 22(12). 301-10. 2010.
[48]   | Jedrejko, K., Kała, K., Ziaja, K.S., Krakowska,. A, Zieba, P., Marzec, K., et al., Improvement of sperm production in subfertile boars by Cordyceps militaris supplement, Antioxidants, 11. 1861. 2022.