Alternative Approaches to Wound Healing

The history of wound healing across the globe abounds with usage of various herbs for treating simple cuts and bruises to serious burns. Wound healing is a complex and dynamic process and, moreover, depends a lot on the wound bearing person’s immunity and mental status. Synthetic medicine may give rise to side effects of allergy and resistance with usually higher cost of treatment. Whereas the alternative and comple‐ mentary medicine such as Ayurveda, Siddha, Unani, Chinese medicine, and ozone therapy can lessen these side effects considerably and offer treatment at lower costs, thus elevating the overall quality of life of the patient. In today’s times the patient is more demanding and has the ability to partake in treatment decisions. It is then the moral responsibility of the scientists to apply modern up‐to‐date scientific acumen to provide evidenced‐based concept to alternative therapies of wound healing to ensure that these practices are safe and efficacious.


Introduction
Wound healing continues to pose a challenging clinical problem despite scientific developments in the field. A correct and efficient wound management is essential. Emphasis is required on new and alternative therapeutic approaches and development of technologies for acute and chronic wound management. A wound can be defined as a damage or a disruption to the normal anatomical structure and function [1]. This can range from a simple break in the epithelial integrity of the skin or it can be deeper, extending into subcutaneous tissue with damage to other structures such as tendons, muscles, vessels, nerves, parenchymal organs, and even bones [2]. Depending on the time of repair, wounds can be divided as acute, chronic, and complicated. Wound healing is a natural, complex, dynamic yet continuous process, and is initiated as any injury occurs and continues till the entire repair of the wound and tissue remodelling is complete. The process can be randomly divided into (i) coagulation and haemostasis, (ii) inflammation, (iii) proliferation, and (iv) wound remodelling with scar tissue formation. If the wound healing is interrupted by any infection, tissue hypoxia, edema, growth factor imbalance or nutritional, and metabolic status of the host, then the wound may take extended time for repair. Normal wound healing is a dynamic and complex process involving a series of coordinated events including bleeding, coagulation, initiation of an acute inflammatory response to the initial injury, regeneration, migration, and proliferation of connective tissue and parenchyma cells, as well as synthesis of extracellular matrix proteins, remodelling of new parenchyma, connective tissue, and collagen deposition [3][4][5][6][7]. Modern synthetic allopathy-based medicines have their share of limitations of allergy, resistance, cost, etc., which has prompted the scientists and wound care professionals to consider alternative approaches to wound healing and validating their use using modern technology. The perception toward alternative medicine such as Ayurveda, Siddha, Unani, and Chinese has changed. The World Health Organisation (WHO) defines traditional medicine as "the sum total of the knowledge, skills, ands practices based on the theories, beliefs, and experiences indigenous to different cultures, whether explicable or not, used in the maintenance of health as well as in the prevention, diagnosis, improvement or treatment of physical and mental illness" [8]. Each of the above-mentioned systems of medical practice offers a variety of medicines for wound management and has been traditionally practiced since ancient times in few parts of the world and is fast getting acceptance in rest of the globe. Ayurveda or Indian traditional and Chinese medicine have rich abundance of knowledge and have been well known as ethnopharmacological and folklore-based systems. Siddha also has origin in south of India, Tamilnadu, which aims in providing ultimate cure to both mind and body systems. "Food as medicine" is the principle behind Siddha treatment. Unani system of medicine has its origin in Iran and also has documented evidences of antimicrobial herbs possessing wound healing properties. Ozone therapy has been in use since World War I times and offers remarkable bactericidal action. The abovementioned alternative therapies offer an economical approach to wound healing. Besides this benefit, they have significant lesser side effects thus increasing patient compatibility as compared to modern medicine.

Ayurvedic products
A rich heritage of knowledge on preventive and curative medicines is available in ancient scholastic work included in the Atharvaveda (an Indian religious book), Ayurveda (Indian traditional system of medicine), etc. Many Ayurvedic plants have a very important role in the process of healing of wounds (vrana). Plants are more potent healers because they promote the repair mechanisms in the natural way. Plant-based therapy not only accelerate healing process, moreover, also maintain the esthetics. More than 70% of wound healing Ayurveda-based pharma products are plant-based, 20% are mineral-based, and remaining contain animal products as their base material. The plant-based materials are used as first aid-antiseptic coagulants and wound wash [9]. Different Ayurvedic preparations are made and used topically for healing of wounds. Ghee is used in many Ayurvedic traditional preparations and also finds use as an ointment base. It is rich source of essential fatty acids (EFAs), which regulate prostaglandin synthesis and hence induce wound healing. Cow ghee (Goghrita) specifically possesses regenerative properties promoting the growth of healthy cells and is clinically proven. Bhasma is a calcined preparation in which the gem or metal is converted into ash. Grithas, also called as neyyu, are medicated clarified butter. Taila or medicated oil is manufactured by steeping powdered medicinal substances, water, vegetable drugs in paste form and fragrance-producing materials such as cardamon, saffron, sandalwood, camphor. Malam (Cream) too is applied topically and has been documented in ancient ayurvedic texts for its helpfulness in wound healing. Some of the herbal drugs that are mentioned in Ayurvedic texts which have been specifically studied for their wound healing properties (vranaropaka) are mentioned below.

Aloe vera
A. vera finds a mention in Ayurvedic practice since centuries regarding its wound healing property. A study carried out by Yadav et al. [10] in 2012 provides the scientific rationale for the traditional use of A. vera gel for management of wound using wound excision model in experimental rats. The effect produced by A. vera gel with reference to wound contraction, wound closure, decrease in surface area of wound, tissue regeneration at the wound site, and histopathological characteristics were significant in treated rats. The effect of A. vera gel on biochemical studies revealed significant increase in collagen and decreased hexosamine content and malondialdehyde levels when compared with control. The authors concluded that A. vera gel is very effective on open wounds and a promising herbal drug. It also had a marked influence on the collagen level which is the precursor protein for wound healing mechanism. A. vera gel reportedly accelerated epithelialization, neovascularization, and increased wound contraction in the later stage of the wound healing process.

Cleome rutidosperma DC.
A study [11] justifies the use of folklore plant C. rutidosperma for the treatment of wounds. Petroleum ether, chloroform, methanol, and aqueous extracts of C. rutidosperma (Family: Capparidaceae) roots were evaluated for their wound healing activities in rats using excision and incision wound models, respectively. The effects of wound healing were assessed by the rate of wound closure, period of epithelialization, and wound breaking strength. Nitrofurazone (0.2% w/w) in simple ointment IP was used as reference standard for the activity comparison. The authors concluded that the animals treated with methanol and aqueous extracts of C. rutidosperma showed faster rate of wound healing compared with other extracts under study. The wound healing property of the roots can be attributed to the presence of flavonoids, triterpenoids, and tannins which possess the antimicrobial and astringent properties which seem to be responsible for wound contraction and increased rate of epithelialization.

Kshatantak Malam
It is a combination of Acyranthes aspera, Allium cepa, and Cannabis sativa also known as Baharer nani has been traditionally reported by Bengal School of Ayurvedic Physicians as external healer for open cuts, complicated wounds and burns but still a scientific document proving its quality was not available. Gangopadhyay et al. [12] reported its pharmacological evaluation and chemical standardization for its wound healing activity in rats. The test drug was applied topically on a 8 mm diameter full thickness punch in Wistar rats. Framycetin and povidoneiodine ointment were used as standard comparators. Parameters such as wound contraction size (mm 2 ), wound index, healing period (days), tensile strength (g), DNA (mg/g), RNA (mg/ g), total protein (mg/g), hydroxyproline (mg/g), PAGE study, and histological analysis were carried out for analysing the effects of the malam. Out of the three constituents, A. aspera specifically possesses potent wound healing activity, A. cepa owns antimicrobial activity, and C. sativa is capable of tissue repair by virtue of its anti-inflammatory property.

Katupila (Securinega leucopyrus) (Willd.)
Another plant used since ancient times in Saurashtra region of India and Srilanka is known as Katupila in Srilanka and Humari in India. It is mentioned as one of the 60 measures for wound healing by Acharya Sushruta (Ancient spiritual Hindu teacher). Katupila leaves act as antiseptic, and the paste is capable of removing extraneous material from the wounds without the need of surgery [13].

Ayurvedic polyherbal formulation
Rawat and Gupta [14] reported wound healing activity of a prepared ayurvedic formulation containing Jasad Bhasma, Gandhak, Tankankhar, and Ras Kapoor. J. (Yashad) Bhasma is ash containing zinc. Gandhak or Gandhaka Rasayan is an ayurvedic mineral-based medicine, which contains detoxified sulfur processed with herbal juice as a main ingredient. Gandhak Rasayan is a great antibacterial, antiviral, and antimicrobial ayurvedic medicine. Tankankhar is borax applied topically for its analgesic property. Ras Kapoor are pills made out of camphor (karpoora/ kapoor), mercury compound (Shuddha Hingula), purified opium (Papaver somniferum), nutgrass (Musta/ Cypeus rotundus), Connessi seed (Indrayava/ Holarrhena antidysenterica), and Nutmeg (Jatiphala/ Myristica fragrans). The preparation was studied on excision and incision wound models in rats. The preparation exhibited remarkable wound healing in rats and the authors suggest further mechanism-based probing to prove the effectiveness of the constituents to be utilised in humans.
Few more folklore plants which were validated by means of preclinical wound healing studies are mentioned below in Table 1. Almost all the plants are shown to be wound healers by applying their extracts topically on wounds created by punches on rats. Their subsequent effect on wound healing models such as excision, incision, and dead space wound models in rats corroborate their wound healing properties. These plants ultimately are reported to stimulate wound contraction, increase hydroxyproline and eventually collagen content thus strengthening the wound area, increase the wound closure rate, reduce scar area, and epithelialization period. Few plants such as A. cepa, Ageratum conyzoides, and Heliotropium indicum are known to specifically heal the wounds by benefit of their antioxidant property. Dexamethasone is a potent anti-inflammatory glucocorticoid which is used in skin allografts but is known to delay wound healing. Plants such as Ocimum sanctum, Gossypium herbaceum, Ficus hispida, Pyrus communis, and Tetrapleura tetraptera have been specifically shown to promote dexamethasone suppressed wound healing tested in wound healing models ( Table 1). Alafia multiflora is found to be one of the plants which contains retinoids and have shown marked interaction with steroids like dexamethasone and was reported to further delay the healing of dexamethasonesuppressed wounds. Thus, vitamin A even if known to hasten the process of wound healing is shown to interact with steroids and thus concomitant application should be avoided in dexamethasone suppressed wounds. Few plants such as Echinacea species, Vitamins such as A and E found in plants, Bromelain, and Grape seed extract have demonstrated few interactions with conventional therapies used for wound healing (

5.
Piper betle (Piperaceae) In Indian folkloric medicine, betel leaf is popular as an antiseptic and is commonly applied on wounds and lesions for its healing effects. This particular property has paved way for further experimental studies, which have established pan extract to have antimicrobial and anti-leshmian properties. Fresh juice of betel leaves is also used in many Ayurvedic preparations [19] 6.
Moringa oleifera (Linn.) It has anti-inflammatory, antibacterial and counter irritant action, which helps in wound healing. The aqueous extract was studied and it was found that there was It is used for cuts, wounds and to boost the immune system [21] 8. Terminalia Chebula Traditionally, fruit pulp is used to stop bleeding. Recent studies showed that various aqueous and organic extracts increased cell proliferation and reduced free radical production thus promoting wound healing. Significant utility of the plant is in cases where ammonia accumulation is a limiting factor [22] 9.
Aegel marmelos (Bael) (Rutaceae) Methanolic extract was found to heal wounds faster when tested in wound healing models in rats. A. marmelos treated wounds were reported to epithelialize faster and rate of wound contraction was higher as compared to control group of rats [23] 10. Alternanthera sessilis Consist of chemical constituents like α and β-spinosterols lupeal isolated from roots.
The leaves are used for cuts and wounds. The wound healing property of A. sessilis (Linn.) attributed to sterols present in the plant [24] 11. Mussaenda frondosa.
The leaves extract was tested on wound healing models in rats. M. frondosa treated rats displayed Increased wound concentration and tensile strength, augmented hydroxyproline content along with antibacterial activity [25] 12. Aristolochia bracteata (Aristolochiaceae) and Cassia tora (Leguminosae) Wound contracting ability of the extracts was significantly greater than that of the control, which was comparable to that of the reference standard 0.02%w/w nitrofurazone ointment [26] 13. Mimosa pudica 14. Anthocephalus Cadamba The potent wound healing capacity was shown from the wound contraction and increased tensile strength has thus validated the ethno therapeutic claim [28] 15. Lantana camara (Linn.) Showed considerable signs of dermal healing and significantly decrease mean wound healing time and reduced scarring at the wound enclosure [29] 16. Carapa guianensis The ethanolic leaf extract of C. guianensis showed increase in the rate of wound contraction, skin breaking strength, the rate of epithelialization [30] 17. Curcuma longa (Linn.)  The stem bark aqueous extract was studied for effect on dexamethasone delayed wounds in excision and incision wound models in rats. The authors reported excellent potential of the plant related to epithelialization, contraction and tensile strength improvement [42] Plant reported to display interaction with dexamethasone and further delay wound healing (drug-herb interaction) 29. Alafia multiflora (Stapf.) The study on A. multiflora highlights the drug-herb interaction. Aqueous extract of the plant was studied for its effect on normal and dexamethasone delayed wounds in rats utilizing excision and incision wound models. It showed beneficial effects in normal rats. But in dexamethasone delayed wounds, the plant extract further deteriorated the wound healing. This proved the known interaction between retinoids occurring in the plant and the steroid but also indicated at involvement of some other constituent contributing to the delayed wound healing [43] Plants with interactions with conventional therapies and reported side effects

Siddha medicines
Siddha is the traditional system of medicine practiced in Tamilnadu, South India, and Tamil speaking areas of the world. The Siddhars' (founders of Siddha medicine) used lots of herbs to treat cardiac and respiratory conditions, heal wounds, treat snake and scorpion bites, dreaded diseases like cancer, and documented them in the form of palm leaf manuscripts, stone, and copper scriptures, etc. The claim of the Siddha physicians is that the medicinal plants are commonly identifiable, easily available throughout the year, cost effective, and with lesser adverse effects [49]. The Siddha system of medicine aims at offering complete cure to mind and body. It follows the principle of "Food as medicine" [50]. The Siddhars have documented many oils (Tailam) and ointments (Kalimbu) effective in wound healing. There are few plants mentioned in Siddha system of traditional medicine which have been evaluated preclinically for their wound healing activity.
Few noteworthy studies using Siddha medicines are mentioned below.

Siddha polyherbal oils
Siddha medicine has used Kayathirumeniennai, punguthailam, and mathanthailam as polyherbal (medicated oils) since centuries, but recently, Sabarianandh et al. in 2014 made efforts to scientifically validate the use of these oils on experimentally induced burn wounds in Wistar rats. The study showed wound contraction was significantly decreased; epithelialization was increased as compared to the vehicle treated group. This was even confirmed by the histopathological analysis [51].

Heritage Sanjeevi
Pugalendhi et al. demonstrated the effect of Heritage Sanjeevi (a Siddha combination drug) on wound healing in Wistar rats. "Heritage Sanjeevi" is a medication made up of Curcuma Aromatia, Psoralea Corylifolia, Vernonia Anthemintica (Willd.), Hydnicarpus Laurifolia, Elettaria Cadamomum, coconut milk, mercury, sulphur, hydrogyrum schloride calomel, "Sulphie" of lead, copper sulphate, zinc sulphate, and camphor. The mixtures of these compounds were processed in the manner that all the inorganic material is detoxified using an ancient detoxin called Pooneer. The extracted oil is filtered and stored in glassware. It is used for external application only, and its shelf life is six years, and it has been claimed to have excellent healing property against burns, scalds, chemical burns, acid burns, and radiation burns. The medica-tion was tested on Wistar rats by external application till the time the wound which was created using a punch was completely healed. The study results demonstrated significant reduction in the size of the wound. The authors claimed that wound healing can be credited to one or few constituents of the medication causing collagen production and thus helping in faster wound correction [52].

Polyherbal formulation
Krishnamoorty et al. in 2012 reported an in vitro study of a polyherbal formulation comprising of extracts of Wrightia tinctoria, Aloe vera, Curcuma longa, and Terminalia chebula. They studied the impact of the formulation on fibroblast cell migration and proliferation using scratch wound assay technique. Fibroblast cell migration and proliferation were studied employing cell migration assay. Carbomer-based gel with beeswax made for a novel delivery system and the formulation proved significantly effective in management of superficial wounds and first degree burns [54].

Kungiliya vennai and Kalchunna thailam
Vennai (Butter) and thailam (Balm) are mentioned and used in Siddha medicine as wound healing bases. Kungiliya vennai is herbal formulation containing Shorea robusta, Sesamum indicum, and Cocos nucifera. It is traditionally recommended by Siddha practitioners for wound healing.
Kalchunna thailam finds a mention in Siddha medicine for wound healing and is a preparation of limestone and coconut oil mixed in equal proportions. In an excision wound model in Sprague Dawley rats, the Kungiliya vennai and Kalchunna thailam treated rats showed positive outcome in the wound healing process. The preparations were comparable with the standard 2% Mupirocin ointment. The authors reported Kungiliya vennai has additional property of regenerating adnexal structures such as hair follicles, sweat, and sebaceous glands [55].
Few more Siddha plants which were preclinically evaluated for wound healing activity are mentioned below ( Table 2). Alcoholic Bark extract resulted in a significant decline in the wound in rats. When compared with standard drug, nitrofurazone, it was found superior in terms of wound contracting ability, wound closure time, and tensile strength [63] 9.

coromandelica)
Ethanol and acetone extracts of barks were applied to male Wistar rats in the form of simple ointments exhibited wound healing activity in excision and incision methods. Framycetin sulphate was taken as standard control. It displayed potent wound healing activity in terms of significant wound contraction and increased tensile strength [64] 10. Plantain tree (Musa

marsupium)
The effect of heart wood extract of P. marsupium on wound healing has been studied in diabetic and normal animals. The effect has also been compared with standard (mupirocin ointment) application. The results show that application of heart wood extract significantly increased wound healing in both normal and diabetic animals [66]

Unani medicines
Unani system of medicine has its origin in Iran and also has documented evidences of antimicrobial herbs possessing wound healing properties. Like Siddha, Unani system too has a mention of cow's ghee (Roghan-e-gao), Shorea robusta, etc. Following are examples of wound healing plants or medicines (marham) according to Unani system of medicine.

Iranian wound healing plants
Pirbalouti et al. reported the wound healing properties of five traditional Iranian plants on alloxan-induced diabetic wounds in rats. Wound area, epithelialization time, and histopathological characteristics were studied upon treatment with Malva sylvestris, Punica granatum, Amygdalus communis, Arnebia euchroma, and Scrophularia deserti. The results corroborated the traditional wound healing use of the above plants [67].

Marham-e-Ral
Similarly, a study described the wound healing effect of a Unani formulation Marham-e-Ral in rats. Ingredients of this formulation are Shorea robusta Gaertn (Ral), Camphor (Kafoor), Catechu (Katha), Roghan-e-Gao, and Beeswax. Marham-e-Ral was administered topically once a day till complete epithelialization occurred. Wound contraction and epithelialization were measured. Both excision and incision wound models were developed. Rate of wound contraction was significantly enhanced so was the epithelialization period. The plant extracts have revealed presence of flavonoids, triterpenoids, and tannins which are known to contribute to wound healing. They also possess astringent, antimicrobial and antiinflammatory effects. Based on their study, the authors claimed the prohealing stroke Marham-e-Ral possesses [68].

Aloe (Elva)
Aloe is one of the oldest plants documented across the globe and also features in Unani system of medicine where it is called as 'Elva'. Oryan et al. [69] reported the detailed account of wound healing activity of A. vera in rats. They demonstrated that the wound healing was 50% faster with aqueous extract of A. vera as compared to silver sulfadiazine in case of open wounds.

Golnar-e-farsi
Punica granatum (Linn.), known as Golnar-e-farsi in Iran, popularly known as pomegranate flowers and Achillea kellalensis (Bioss.) and Hausskn a well known traditional herb used in tribal medicine of Iran is locally known as Golberrenjas or Bumadaran-e-Sabzekoh were studied scientifically by Pirbalouti et al. in 2010. The authors reported their significant wound healing properties in rat excision wound model owing to their increased wound contraction ability and collagen turnover [70].

Shorea robusta
Shorea robusta finds a mention in both Siddha and Unani systems of medicine. A study carried out by Wani et al. in 2011 reported the wound healing property of S. robusta Gaertn. f. in excisional and incisional wound models in rats. The ethanolic extract was found to accelerate wound contraction, increased tensile strength, and hydroxyproline content thus acting as a wound healer [71].

Chinese herbs
As are Ayurveda and Siddha to India and Unani to Iran, traditional Chinese system of medicine has been practiced for ages in China. Traditional Chinese Medicine (TCM) focuses on the concept of Yin and Yang which describes two opposing yet complementary aspects of any one phenomenon. Yin is associated with poor circulation and healing and stagnation, while Yang is related with being overheated and excess of scar tissue. Thus for optimum wound healing, an ideal balance needs to be struck between Yin and Yang. Raw Chinese formulas are created specific to each patient. A raw formula means simply that the herbs in the formula are in their natural state without modification. Traditionally, raw Chinese formulas are given in their organic form, cooked for several hours, and then ingested, but it tends to have an undesirable taste. However, with wound healing, a topical application is convenient, effective, and has no or less side effects. Herbs can be utilized in their natural state and with their maximum potency. Few of the examples of wound healer herbs of TCM are as follows:

1.
Tam et al. [72] were the first to report a combination of Radix astragali and Radix rehmanniae in the ratio of 2:1 for the treatment of diabetic foot wounds. The herb combination showed its effectiveness in treating diabetic wound healing through the actions of tissue regeneration, angiogenesis and anti-inflammation.

2.
Angelica sinensis (AS) called as Dang-Gui in Chinese, was analysed in detail by Hsiao et al. [73] using proteomics to demonstrate range of pharmacological effects associated with AS which will prove fruitful in developing it as a wound healing herb. The authors concluded that AS extract and its active component ferulic acid (FA) participate in the modulation of wound healing process associated with fibroblasts. FA specifically acts as a ROS scavenger. Additionally, FA is also able to trigger proteins like heme oxygenase-1 (HO-1), heat shock protein 70 (HSP70), Extracellular signal-regulated kinases (ERK ½), and Protein kinase B (Akt), which help cells to respond to environmental stress thus contributing to its enhanced wound healing ability.

3.
Hou et al. [74] recently demonstrated the wound healing property of a four-herb Chinese medicine ANBP which is a pulverized mixture of four herbs including Agrimonia eupatoria (A), Nelumbo nucifera Gaertn (N), Boswellia carteri (B), and Pollen Typhae Angustifoliae (P) explored the effect of four-herb Chinese medicine ANBP. The herb was evaluated on the basis of wound healing and scar formation in rabbit ear hypertrophic scar models of fullthickness skin defect. Compared with the control group, local ANBP treatment not only significantly improved wound healing, but also reduced scar formation. The study results demonstrated that ANBP treatment along with reducing collagen synthesis, blocked excessive deposition of collagen and also promoted collagen maturity, thus obstructing the formation of scar. The mechanism of the effect of ANBP on collagen expression is different in the early and late stages of wound healing, which is favourable for wound closure and scar contraction. Using proteomics approach, the authors suggested that ANBP promoted wound healing and condensed scarring by bidirectional regulation of the Transforming Growth Factor β (TGF-β)-/Smad-dependent pathway.

4.
Chinese medicine book, Compendium of Materia Medica proclaims Lucilia sericata known as 'WuGuChong' in Chinese for treating superficial purulent diseases like carbuncle. Zhang et al. reported that fatty acid extracts of Lucilia sericata can promote murine cutaneous wound healing by virtue of its remarkable angiogenic activity. Wound excision model in rats followed by Vascular Endothelial Growth factor (VEGF) expression analysis by western blotting, RT-PCR and immunohistochemistry strongly suggested the mechanism involved in wound healing property of the herb [75].

5.
Chak et al. [76] demonstrated the effect of another Chinese medicine Shiunko consisting of sesame oil, Lithospermi radix, (LR; Lithospermum erythrorhizon Sieb. et Zucc.), A. sinensis, lard, and beeswax. The authors reported that Shiunko-treated fibroblasts induced range of biochemical events engaged in the wound healing process, including cell proliferation and anti-apoptosis, anti-oxidant activity, secretion of collagen, and cell mobility. It was also noted that Stathmin, a differentiation marker was greatly induced by Shiunko, which is a sign of good healing process. Proteomics suggested peroxiredoxin and glutathione Stransferase were involved in antioxidantion offered by Shiunko. Also, superoxide dismutase was enhanced after Shiunko treatment which again contributed to its wound healing property. TGF-β was upregulated on Shiunko treatment which happens to be upstream regulator of collagen expression and an indispensable factor for wound healing.

6.
Terminalia chebula is mentioned in other systems of medicine like Ayurveda for its wound healing ability. Likewise, it was proven by Li et al. [77] that tannin extracts from immature Terminalia chebula fruits helps in cutaneous wound healing in rats. The immunohistochemical, transcriptional and translational levels of VEGF analysed in the study helped the authors conclude that the wound healing property was by the virtue of anti-angiogenic effects. Also, the proliferation of bacteria like Staphylococcus aureaus and Klebsiella pneumonia were inhibited by the extract thus conferring the much needed antibacterial effect beneficial for faster wound healing. Thus the study concluded that tannin extracts from immature fruits of Terminalia chebula Fructus (Retz.) stimulated cutaneous wound healing in rats.

Ozone therapy
Ozone therapy dates back to the year 1914 when it was used during World War I for the treatment of gas gangrene. Ozone has multiple therapeutic effects in wound healing due to the property of releasing nascent oxygen, which has been shown to have bactericidal capabilities and to stimulate antioxidant enzymes. There are few randomized clinical trials to verify the use of ozone therapy in the early stages of wound healing. To verify the same, Zhang et al. [78] recently carried out a study assessing the use of ozone therapy in the early stages of diabetic foot ulcer by estimating the expression of VEGF, TGF-β, and Platelet derived growth factor (PDGF). The authors claimed that the oxygen-ozone therapy increased the levels of all the above three endogenous growth factors which contributed to its enhanced wound healing ability. Similarly, Wainstain et al. [79] demonstarted that oxygen-ozone therapy along with conventional therapy for 24 weeks hastened the healing of diabetic foot ulcer. The theory that ozonated oil has wound healing property was investigated at our laboratory in an excision wound model using Sprague Dawley rats. The animals were divided into four groups, which were treated with sesame oil (vehicle), framycetin (standard), or two doses of ozonated sesame oil (peroxide values 500 and 700 mEq/1000 g, respectively). The formulations were topically applied on the excision wounds once daily for 11 consecutive days, and the animals were euthanized on the 12th day. Ozonated oil treated wounds had significantly higher tensile strength, collagen content, and superoxide dismutase activity than that of the vehicle treated wounds. Histopathological analysis of skin of the excised wound area treated with ozonated oil revealed better healing activity in comparison with the vehicle-treated wounds. Thus it was concluded that ozonated oil can be of potential remedial use for healing wounds [80]. Another animal study was carried out by Kim et al. [81] to evaluate the therapeutic effects of ozonated olive oil in guinea pigs in acute cutaneous wound healing model. Full thickness punch wound was created on the back of guinea pigs and ozonated olive oil treatment was compared with pure olive oil and no treatment control group. The immunohistopathological results demonstrated that topical application of ozonated olive oil increased the levels of VEGF, TGF-β, and PDGF thus accelerating acute cutaneous wound healing. A study conducted by Travagli et al. [82] reported a deleterious effect of ozone treatment on aged mice. The authors claimed that ozone therapy in 8-week mice enhanced wound healing, while when administered to 18-week mice, the full thickness excisional wound displayed delayed healing. This may also be attributed to reduced bacterial infection and/or increased O 2 tension by O 3 contact in wound area in younger population.

Clinical studies supporting the folklore use of alternative therapies
Scientists advocate more clinical studies to be conducted to provide robust proof-of-concept of folklore wound healing property of the herbs mentioned in alternative therapies. Few of the clinical studies carried out are documented subsequently.

Katupila study
A case study of diabetic wound of a 55-year-old female patient wherein Katupila paste [(Securinega leucopyrus) (Willd.)] was applied daily and after a month the wound was reported to have healed completely leaving a minimal scar. The healing properties of Katupila were attributed to its antimicrobial, antiseptic, and wormicidal qualities. Also it was shown to possess abundant quantities of flavonoids and tannins which offer the antioxidant effects [13].

Manjishthadi Gritha
A clinical study employing Manjishthadi Gritha was carried out by Baria

Honey: A pilot study
Vijaya et al. [84] reported a pilot study using honey for healing the cutaneous wounds of 10 randomly selected patients of both sexes. Honey collected locally was applied daily for 20 days and the size of the wound was measured on day 7, 15, 20, and after complete wound healing. The authors concluded that honey was remarkable in healing the wounds and can be very effectively used as first aid dressing material. A case study [85] was reported employing honey for the cure of a chronic infected wound on the right lower limb of a 70-year-old female. Every morning the wound was cleaned with neem bark decoction, and Dabur® honey was applied on the wound. Along with the local application, following drugs were administered orally every 12 hours: Glycerrhiza glabra (Linn.), Asparagus racemosus (Willd.), Tribulus terrestris, Tinispora cordifolia (Willd). At the end of fifth week, the authors reported a complete healing of the wound leaving a minimal scar. Sushruta Samhita has mentioned honey (madhu) as a wound healer centuries ago. The above case study practically proved the effectiveness of honey. It is hyperosmolar so confers antibacterial effect. It has high viscosity so acts as physical barrier. Presence of enzyme catalase in honey gives it antioxidant properties. The four drugs given orally possess antioxidant, adaptogenic, and immunomodulatory activities. Clinical studies employing honey with bigger population although would yield more authentic conclusions.

Dragon's blood cream
Namjoyan et al. [86] reported a clinical study using Dragon's blood cream, a deep red resin obtained from four different sources, Croton spp., Dracaena spp., Daemonorops spp., and Pterocarpus spp. 60 patients referred to remove their skin tags were included in this randomized clinical trial to receive either Dragon's blood cream or placebo cream. Wound measurement and process of healing was checked on 3rd, 5th, 7th, 10th, 14th, and 20th day of the trial. The patients receiving Dragon's blood cream showed significant wound healing. The phenolic compounds present in Dragon's blood cream reportedly contribute to its effective wound healing property.

Adjuvant ozone therapy
Shah et al. [87] presented a case study of a 59-year-old female patient with an extensively infected wound and exposed tibia to about 4 / 5 th of its extent. Topical ozone therapy twice a day and ozone hemotherapy once a day along with daily dressings and parenteral antibiotics showed significant improvement in 15 days in the patient. After a follow-up of 20 months, the patient was able to walk with minimal disability. Ozone disintegrates into reactive oxygen species which further lead to increased growth factors contributing to faster wound healing.
The study stated that oxidative injury is a possible side effect of ozone therapy but at therapeutic doses oxygen radicals are removed by the blood antioxidants and thus side effects are rare and only observed in overdose or compromised anti-oxidant system.

Conclusion
The folklore knowledge is abundant with mention of various herbs and medicines as wound healers across different civilizations and systems of medicines. Modern medicine is fast attempting to explore the ancestral data and test its effectiveness using current experimental methods. The focus is on analyzing the molecular basis of the effectiveness of the concerned plants. Proteomic and genomic-based evidences using robust markers blended with traditional knowledge can provide relevant answers and solutions for healing of wounds which affect the largest organ of human body. Although more randomized clinical trials are the need of the day to validate the ancient claims.

Author details
Anuradha Majumdar * and Prajakta Sangole *Address all correspondence to: anuradha.majumdar@bcp.edu.in Department of Pharmacology, Bombay College of Pharmacy, Kalina, Mumbai, India