Evaluation of the wound healing activity of twelve herbal and non-herbal remedies used in Sana’a-Yemen for the treatment of wounds and burns

Wound healing is a complex process of integrated and overlapping phases resulting in the restoration of structural and functional integrity of injured tissues. Wounds and burn wounds represent a significant burden on the patients and health care professionals worldwide. There is still an obvious lack of suitable wound healing drugs since most of the available products can have some side effects and limitations. Traditional medicine has been practiced by the indigenous people of Yemen from antiquity until today. However, many of herbal and non-herbal products, used for wounds treatment, have not been the subject of any scientific investigation. Hence, the aim of this study was to document and scientifically evaluate the claimed wound healing efficiency of twelve herbal and non-herbal remedies (nine plants, one mineral (potassium alum), one marine product (cuttlefish bone) and one animal product (honey) used in Sana’a by indigenous people to treat wounds and burns. Searching electronic databases has indicated various experimental studies demonstrating four distinct pharmacological activities (anti-inflammatory, antioxidant, antimicrobial activities and promoting various phases of wound healing) displayed by the raw materials, extracts, chemical groups and some isolated compounds of the studied herbal and non-herbal remedies. According to the reviewed literature no serious side effects have been described for the remedies when applied topically. This study provides scientific data that support the wound healing efficiency of the studied remedies and thus lend some scientific justification for their traditional use in Sana’a for the treatment of wounds and burns. These herbal and non-herbal remedies can be considered as leads for future materials for wound healing and therefore further experimental and clinical studies are required to validate their effectiveness and safety.


INTRODUCTION
Traditional medicine (TM) is widespread throughout the world and has been practiced for centuries. According to WHO [1], the use of TM has increased, and more countries are recognizing the role of traditional and complementary medicine in their national health systems. In Yemen, traditional medicine remains prevalent in rural areas and to some extent in urban areas. The wider acceptance of herbal medicine among Yemeni people is due to several reasons such as rich flora, better accessibility, poverty, low cost of herbal drugs, shortage of hospitals and health centers in remote areas, and lack of faith in modern medicine. Yemeni medicinal herbal and non-herbal materials have been used by indigenous people for the treatment of a number diseases such as infectious, parasitic, pulmonary (including tuberculosis), gastrointestinal, urogenital, ocular and skin diseases, including wounds and burns [2][3][4][5][6].
Wounds are major causes of physical disabilities. According to the wound healing society, wounds are physical injuries that result in an opening or breaking of the skin that causes disturbance in the normal skin anatomy and function [7]. Wounds represent a significant burden on the patients and health care professionals worldwide. They not only affect physical and mental health of millions of patients but also impose significant costs on them. Current estimates indicate that worldwide nearly 6 million people suffer from chronic wounds. Unhealed wounds constantly produce inflammatory mediators that produce pain and swelling at the wound site. Chronic wounds may even lead to sepsis with multiple organ failure or death of the patient [8]. According to the WHO's Evaluation of the wound healing activity of twelve herbal and non-herbal remedies used in Sana'a-Yemen for the treatment of wounds and burns Alasbahi and Groot International Classification of Diseases version 10 (ICD-10), burn injuries are classified by site of injury in chapter XIX as "burns and corrosions", (T20-T32) and in terms of aetiology they are classified as those caused by exposure to smoke, fire and flames (X00-X09), contact with heat and hot substances (X10-X19), exposure to electric current (W85-87), lightening (X33) and exposure to corrosive substances (X46-X49). Therefore, burns include scalds as wells as injuries caused by heat from electrical heating appliances, electricity, flame, friction, hot air and hot gases, hot objects, lightening and chemical burns (both external and internal corrosions from caustic chemicals). Radiation-related disorders of the skin and subcutaneous tissue and sunburn are not included in this classification of burns [9]. According to the depth, burn wounds are classified as first degree (superficial), second degree (partial thickness) and third degree (full thickness) [10]. Burns are a global public health problem, accounting for an estimated 180 000 deaths annually. The majority of these occur in low-and middle-income countries and almost two thirds occur in the African and South-East Asia regions [11].
Wound healing represents a dynamic and complicated process involving a series of co-ordinated events, including haemostasis, inflammation, proliferation, and tissue remodelling resulting in the repair of severed tissues and restore their structural and functional integrity [10,12]. Although several topical preparations are present on the market for management of wounds and burn-wounds, there is still an obvious lack of suitable drugs since most of the available products have antimicrobial activity rather than a wound healing effect. In addition, they can lead to probable negative effects on healing and even toxicity, as in the case of silver sulfadiazine on fibroblasts [10], neutropenia, methaemoglobinemia and renal toxicity [13,14]. Medicinal plants can act as wound healing agents because of their variety of different constituents like alkaloids, essential oils, flavonoids, tannins, terpenoids, saponins, fatty acids and phenolic compounds, which are potentially able to improve the healing process of wounds. Low cost, availability and fewer side effects are other advantages of herbal remedies [10]. More than 70% of wound healing pharma products are plant based, 20% are mineral based and the remaining contain animal products as their base material [7]. In recent times, focus on plant research has increased all over the world and a large body of evidence has been collected to show the immense potential of medicinal plants used in various traditional systems [7]. Hence, we conducted our work with the objectives to scientifically document and evaluate twelve herbal and non-herbal remedies used in the city of Sana'a (Yemen) for the treatment of wounds and burns. Consequently, we aimed to firstly contribute to preserving the indigenous knowledge of traditional medicine and save it from disappearing as well as secondly, to provide scientific justification for the use of these natural materials as wound healing agents. It is hoped that this study will encourage interest for further scientific investigations regarding the wound healing effectiveness and safety of the remedies's raw materials being used and their active constituents.

Study Area
The study area is the city of Sana'a. This is the largest city in Yemen and the center of Sana'a Governorate. Sana'a has an area of 126 km 2 and a population of approximately 3,937,451 [15]. Detailed information about the vernacular names, uses, preparations and mode of administrations of the herbal and non-herbal remedies used in Sana'a for the treatment of wound and burn wounds were obtained by questioning the traditional medicine practitioners in their shops and practices.

Literature Review
Electronic databases of Leiden University library catalogue were searched for pharmacological activities demonstrated by in vivo, in vitro, or clinical studies and their possible mechanisms of action related to wound healing efficacy as well as the side effects for each of the herbal and non-herbal remedies involved in this study.

RESULTS AND DISCUSSION
Skin disorders, especially wounds and burn wounds, represent a global public health problem as their treatment is very costly and time-consuming. Moreover, the treatment of non-healing wounds, such as diabetic foot ulcers, has always been a challenge. Silver sulfadiazine, an antibacterial agent, introduced as the gold standard in topical burn treatment was found to cause side effects such as delaying the wound healing process [16,17], and toxicity to host cells [18]. Moreover, silver sulfadiazine has systemic complications such as neutropenia, methemoglobinemia and renal toxicity [13,14]. The silver released from its commercial products was reported to cause transient leukopenia secondary to bone marrow suppression [17] and cytotoxic effects on both keratinocytes and fibroblasts [19]. So, there is a need for better and more cost-effective alternatives for the treatment of wounds and burns that are devoid of serious side effects. Plants and other natural products that have been used in traditional medicine for a long time represent a valuable source to be explored for a variety of pharmacologically active secondary metabolites that may help in the treatment of diseases. Based on data collected by questioning the traditional medicine practitioners, we documented twelve herbal and non-herbal remedies used in Sana'a for the treatment of wounds and burns. The herbal materials were identified at the Agriculture Research Center in Dhamar-Yemen. Table 1 presents the ethnobotanical data of the herbal and non-herbal remedies used in Sana'a for the treatment of wounds and burns.
The herbal and non-herbal remedies used in Sana'a for the treatment of wounds and burns includes nine herbs from various families (Asphodelaceae, Compositae, Fabaceae, Lamiaceae, Linaceae, Lythraceae, Myrtaceae, Polygonaceae), one mineral (potassium alum), one marine product (cuttlefish bone) and one animal product (honey). They are prepared in the forms of infusion and decoction for rinsing wounds or used topically either as crushed or powdered materials or as a mixture with fat, or egg white (Table 1). These remedies (Table 1) can be grouped into 1-remedies (such as Aloe vera leaves, honey and Rumex nervosus leaves) used for the healing of both wounds and burns indicating the possibility of a broad spectrum of their pharmacological effectiveness; and, 2-those remedies used either for burn healing only (such as tragacanth gum, cuttlefish bone, Linum usitatissimum seeds, Meriandra bengalensis leaves and Punica granatum fruit peels) or for wound healing only (such as Chamomilla recutita flowers, Myrtus communis leaves, potassium alum and Thymus laevigatus leaves) suggesting a possible selectivity of their effectiveness. It is noteworthy to mention that local herbal healers did not specify the depth of burn wounds they treated.
Searching scientific literature has revealed various experimental studies illuminating different pharmacological properties of extracts and chemical constituents of the studied remedies that may contribute to their wound healing activity. The studied remedies have shown their efficiency in wound healing via four essential pharmacological activities namely, anti-inflammatory, antioxidant, and antimicrobial activities as well as wound healing activity (via modulating one or more of the wound healing phases). Seven remedies (A. vera leaves, C. recutita flowers, cuttlefish bone, honey, L. usitatissimum seeds, M. communis leaves, and P. granatum pericarp) were found to exert their wound healing activity by all four pharmacological activities, whereas the remaining five remedies were found to exhibit their wound healing activity either via three (T. laevigatus leaves), two (M. bengalensis leaves, potassium alum, and R. nervosus leaves) or one (tragacanth gum) pharmacological activities ( Table 2).
In addition, histamin release and formation was inhibted by aloin (70% inhibtion of histamin release comparing with indomethacin (35%) [262], and magnesium lactate [40], respectively. C. recutita flowers aqueous extract was reported to inhibit PGE2 prodiction due to the suppression of the COX2 gene expression and direct inhibition of COX2 enzyme activity [263], while supercritical carbon dioxide extract was found to inhibit 5-LOX, COX and the oxidation of arachidonic acid with IC 50 of 6-25 µg/ml [88]. Several C. recutita flowers constituents were found to inhibit COX and LOX enzymes; chamaviolin and chamazulene carboxylic acid inhibited COX-2 [91], apigenin inhibited 5-and 12-LOX (IC 50 = 8 µM and 90 µM, respectively), chamazulene and (-)-α-bisabolol inhibited 5-LOX (IC 50 = 13 µM and 40 µM, respectively),while apigenin, cis-en-yn-spiroether and (-)-α-bisabolol inhibited COX (IC 50 = 70-80 µM) and both bisabolol and bisabolol oxide inhibited 5-LOX, although bisabolol was the more active of the two compounds [88]. Moreover, α-(-)-bisabolol [94] and matricine [95] were found to inhibit the production of proinflammatory cytokines (tumor necrosis factor (TNF)-α and IL-6) and the nuclear factor kappa B (NF-κB) signalling, respectively. Cuttlefish bone was demonstrated to reduce of the levels of white blood cells, TNF-α, and IL-6 [122]. Honey was reported to reduce the numbers of inflammatory cells present in wounds [129,130]. L. usitatissimum seeds fixed oil was found to inhibit the prostaglandin 2-, leukotriene-, histamine-, bradykinin-, and arachidonic acid-induced inflammation [139]. L. usitatissimum seeds constituents, α-linolenic acid and tocopherol, have been shown to inhibit COX and LOX pathways [140] and to attenuate proinflammatory cytokines and chemokines production [141], respectively. M. communis leaves essential oil, topically applied, was found to inhibit the migration of neutrophils to the inflamed area and the TNF-α and IL-6 formation [155]. In addition, the 80% ethanol leaves extract was reported to reduce the COX-2 and iNOS expression level in lipopolysaccharide (LPS)-stimulated J774A.1 macrophage by 10.50 ± 3.59 fold (P<0.05) and 26.73 ± 3.05 fold (P < 0.01), respectively versus control group [156]. M. communis leaves constituents (myrtucommulone (MC), and semimyrtucommulone (SMC) have been shown to inhibit COX-1 and 5-LOX directly at IC 50 values in the range of 1.8 to 29 µM, and prevent the mobilization of Ca 2+ in polymorphonuclear leukocytes, mediated by G protein signalling pathways at IC 50 values of 0.55 µM and 4.5 µM, respectively, as well as suppress the formation of reactive oxygen species and the release of elastase at comparable concentrations [157]. In addition, MC has been shown to inhibit the microsomal PGE2 synthase-1 and reduced the formation of PGE2 (in three different assays (cell-free, cellular, and whole blood assays at approximately IC 50 values of 1 to 3 µmol·L -1 ) without significant inhibition of the COX enzymes. The SMC also inhibited m P G E S -1 a c t i v i t y, a l t h o u g h l e s s p o t e n t l y (IC 50 = 10 µmol·L-1) [159]. Moreover, MC was found to exert anti-inflammatory activity, in two in vivo models of acute inflammation in mice by decreasing carrageenan-induced paw edema (68% inhibition at 4.5 mg/kg i.p. pretreatment comparing to 57% inhibition by the reference indomethacin at 5mg/kg) and reducing the inflammation in carrageenan-induced pleurisy model (MC ,4.5 mg/kg i.p. 30 min before and after carrageenan, reduced the exudate volume and leukocyte numbers, lung injury and neutrophil infiltration, lung intercellular adhesion molecule-1, P-selectin immunohistochemical localization, TNF-α, IL-1β levels in the pleural exudate and their immunohistochemical localization in the lung, leukotriene B4 level in the pleural exudates, lung peroxidation and nitrotyrosine and poly(ADP-ribose) immunostaining) [158]. Pomegranate rind aqueous extract, applied topically to ex vivo skin, has been demonstrated to downregulate the expression of COX-2 more than the total pomegranate tannins [219,235], while the aqueous ethanol and methanol extracts were found to inhibit the NO production induced by LPS by 67% in comparison to dexamethasone (95%) [195], and the release of endogenous inflammatory mediators [194], respectively. It has been shown that a standardized pomegranate rind extract containing 13% w/w ellagic acid was a potent inhibitor of the NO production [197] and its topical application had an excellent anti-inflammaotry effect due to the inhibition of leukocyte infiltration and blockage of the proinflammatory cytokines [198]. The pomegranate peel constituents, punicalagin, punicalin, strictinin A, and granatin B were found to inhibit NO production and iNOS expression in RAW 264.7 cells. Among them, granatin B showed the strongest iNOS and COX-2 inhibitory effects [200]. In addition, ellagic acid, punicallin and punicalagin were reported to decrease the mRNA expressions of the proinflammatory factors, TNF-α, interferon gamma (IFN-γ) and IL-6, in oxidatively stressed mice [220]. The anti-inflammatory mechanism of action of T. laevigatus leaves has not been yet illustrated. However, the compounds, thymol and carvacrol, were reported to exert anti-inflammatory activities; thymol inactivated calcium channels machinery and thus triggered a corresponding reduction of elastase release [250] and carvacrol inhibited the production of PGE2 catalysed by COX-2 with an IC 50 value of 0.8µM, similar to standard inhibitors indomethacin and NS-398 with IC 50 values of 0.7 µM and 0.8 µM, respectively. The COX-1 was also inhibited by carvacrol approximately at the same rate (IC 50 = 0.7 µM), which suggests non-selective inhibition of both enzyme isoforms [251]. These compounds (carvacrol (84%) and thymol (52.46%) were reported to be the main constituents of the volatile oils obtained from the leaves of the Yemeni T. laevigatus growing in Haggah [252] and Sana'a [253], respectively.
The production of free radicals at or around the wound bed may contribute to delay in wound healing through the destruction of lipids, proteins, collagen, proteoglycan, and hyaluronic acid. Agents that demonstrate a significant antioxidant activity may, therefore, preserve viable tissue and facilitate wound healing [8]. Principal mechanisms applied by antioxidant compounds include the scavenging of free radicals, the reduction of metals such as iron and copper, creating complexes with metal pro-oxidants (chelating), quenching single oxygen, and stimulating antioxidative defense enzymatic activities (decreasing the cellular level of free radicals either by inhibiting the activities or expressions of free radical generating enzymes such as NAD(P)H oxidase and xanthine oxidase or by enhancing the activities and expressions of endogenous antioxidant enzymes such as superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase and increasing cellular antioxidants). The methods used for evaluating the antioxidant activity frequently depend on the reaction mechanisms of the antioxidants. These antioxidants are capable of inactivating radicals with two major mechanisms of hydrogen atom transfer (HAT) and single electron transfer (SET). HATbased methods measure the ability of antioxidants to disable free radicals by hydrogen donating. SET-based methods detect the ability of antioxidants to transfer single electron to reduce any compound, including metals, carbonyls, and radicals [264,265]. Ten of the herbal and non-herbal remedies extracts and/or their chemical constituents ( ), hydroxyl • OH, and alkyl free radicals as well as hydrogen peroxide. In addition, A. vera gel [43], and its ethanol extract [42], the aqueous extract of the leaves (without the gel) [46], as well as the constituents, namely, propyl-7-methoxy-5-methyl-chromone [50], isorabaichromone, and aloeresin B [34], the polysaccharides, APS-1[52], GAPS-1 and SAPS-1[53] as well as aloe-emodin [31,48,49] were found to suppress lipid peroxidation. Other antioxidant activities reported for A. vera extract and constituents were the reduction of the levels of reactive oxygen species in oxidative stressed Vero cells by the polysaccharide APS [54], the ferric reducing antioxidant activity by the methanol extract of leaf skin [47] and the polysaccharides GAPS-1 and SAPS-1 [53], as well as the ferrous chelating effects by GAPS-1 and SAPS-1 [53]. Moreover, A. vera gel [43], its ethanol extract [42,44] and the polysaccharide APS-1 [52] were reported to recover or increase the reduced activities of the antioxidant enzymes such as SOD, CAT, glutathione peroxidase, glutathione-S-transferase and glucose-6-phosphate dehydrogenase as well as glutathione, and uric acid. C. recutita flower extracts, namely, methanol [100], aqueous [102], 70% ethanol [97], ethyl alcohol-water (1:1) [99], 70% aqueous methanol [101], and water, ethanol and methanol [98] extracts as well as the essential oil [103,266] and the constituents of C. recutita flowers viz, the flavonoids (apigenin-7-O-(6"-acetyl)-glucoside, luteolin, apigenin, eupatolitin, and chrysosplenol D) [101], α-bisabolol and chamazulene [106], and α-bisabolol oxide A and (E)-βfarnesene [266] were found to scavenge DPPH • radicals. Polyphenolic-polysaccharide conjugates isolated from flowering parts of C. recutita displayed DPPH • and ABTS +• radicals scavenging activity [108]. Inhibition of lipid peroxidation was demonstrated by dichloromethane extract of the distillate [96], ethyl alcohol-water (1:1) [99], water, ethanol, and methanol [98] extracts, and the essential oil [103] of C. recutita and its constituent chamazulene [89]. Pre-incubation of blood plasma with chamomile polyphenolic-polysaccharide conjugates was also found to diminish significantly the extent of ONOO¯induced oxidative stress of the biomarkers of blood plasma proteins such as protein carbonyl and thiol groups, nitrated proteins, and the formation of lipid hydroperoxides [108]. It has been demonstrated that chamomile water extract [102] showed ferric reducing activity and the polyphenolicpolysaccharide conjugates [108] increased the ferric reducing ability of blood plasma. Moreover, α-bisabolol was reported to inhibit both the reactive oxygen species production during human polymorphonuclear neutrophil bursts [104] and the upregulation of H 2 O 2 -generated free radicals in human skin fibroblasts in vitro [105]. Polysaccharides with a total sugar content of 86.2% extracted from the cuttlebone of Sepia aculeate were found to possess scavenging activity against DPPH • , superoxide and hydroxyl radicals as well as ferrous ion-chelating effect [124]. Cuttlefish bone (CB) ointment (CB extract: white petroleum 4:6) was shown to inhibit lipid peroxidation [122]. Honey treatment of patients with partial-thickness burns was reported to decline the levels of serum lipid peroxide [132]. L. usitatissimum seed hull oil was shown to exhibit DPPH • scavenging activity [143]. Yemeni M. bengalensis leaves essential oil was found to possess very weak DPPH • scavenging activity [151]. Different extracts (methanol, ethanol, ethyl acetate, and water) of M. communis leaves were found active as DPPH • scavengers [156,[161][162][163][164]. Among the constituens (four hydrolyzable tannins (oenothein B , eugeniflorin D2 , and tellimagrandins I and II), two related polyphenolic compounds isolated from M. communis var italica were reported to exhibit DPPH • radical scavenging activities, and potent inhibitory effects on xanthine oxidase activity and its ability to generate the superoxide anion O 2 •-radicals [169]. Anti-lipid peroxidation activity evaluated by using several test systems was ascribed to M. communis leaves 70% ethanol extract, and its fractions (ethyl acetate and aqueous extracts) [160], methanol, ethanol and water extracts [163], and methanol extract [164], essential oil [165], and constituents such as semi-myrtucommulone and myrtucommulone [166,167], myricetin-3-O-galactoside and myricetin-3-O-rhamnoside [169]. Moreover, the ethanol, methnol and water extracts of M. communis leaves were reported to exhibit ferric reducing antioxidant activity [156,[162][163][164]. P. granatum fruit peel extracts, namely, methanol [196,201,217], 75% aqueous methanol [210], ethanol [206,215], water [217] extracts, methanol, ethanol and acetone fractions [208], a mixture of methanol, ethanol, acetone and water extract [204] and ellagic acid rich extract [209] were found to exhibit scavenging activity against one or more of DPPH • , ABTS •+ , hydroxyl, peroxyl and nitric oxide radicals. The active constituent of P. granatum fruit peel, punicalagin, was found able to scavenge ABTS •radicals and repair ABTS •-, guanosine, and tryptophan radicals, generated by pulse radiolysis, via electron transfer [221]. In addition, commercial standard punicalagin was reported to possess DPPH • radicals and H 2 O 2 scavenging activity [222]. Moreover, punicalagin, punicallin and ellagic acid were shown to scavenge DPPH • and O 2 •- [220] and the prodelphinidin dimers (gallocatechin-(4-8)-catechin, catechin-(4-8)-gallocatechin and gallocatechin-(4-8)gallocatechin) were found more potent ABTS •+ radical scavengers than the gallocatechin monomer [218]. Lipid peroxidation was reported to be inhibited by the extracts and some constituents of P. granatum fruit peel as follows: methanol extract [201,202,207], 75% aqueous methanol extract [210], 50% aqueous methanol extract [211], ethanol extract [207,216], acetone and chloroform extracts [207] and a mixture of methanol, ethanol, acetone and water extract [204]; punicalagin [220,221], punicallin and ellagic acid [220] as well as gallocatechin-(4-8)-catechin [218]. Moreover, methanol extract was found to strongly protect LDL from oxidation [201]. Antioxidant activity by increasing endogenous antioxidant enzymes such as SOD, CAT, and gluthathione peroxidase in normal experimental animals was shown by the 50% aqueous methanol extract of P. granatum fruit peel [211], while preserving or increasing of such enzymes in oxidatively stressed experimental animals were demonstrated by the methanol extract [202] and by punicalagin, punicallin and ellagic acid, respectively [220]. The ability to reduce one or more of the metal ions such as iron in the ferric reducing antioxidant power assay, copper in the cupric reducing antioxidant capacity assay or molybdenum in phosphomolybdenum method has been demonstrated by a number of P. granatum fruit peel extracts and constituents such as ethanol extract [215], ethyl acetate extract [212], methanol, ethanol, acetone and ethyl acetate fractions [208], ethyl acetate, acetone, and methanol extracts [203], a mixture of methanol, ethanol, acetone and water extract [204,205], as well as by punicalagin [220,222], ellagic acid and punicallin [220]. Furthermore, the compound punicalagin was found to possess chelating ability toward iron [222]. Yemeni R. nervosus leaves extracts such as the methanol extract [243], crude methanol extract and its sub fractions (ethyl acetate, methanol, n-hexane and chloroform) as well as the oil (containing the methyl esters of palmitoleic acid (28.35%), palmitic acid, (25. 37%) and stearic acid (20.25%) as the major constituents) [245] have been demonstrated to exhibit DPPH • radical scavenging activity. Ethiopian R. nervosus leaves ethyl acetate extract was reported to exhibit DPPH • , ABTS •+ and O 2 •radical scavenging activities, Fe 2+ chelating activity, and ferric reducing antioxidant activity [244]. DPPH • scavenging activity was also shown by the methanol and dichloromthane extracts of Yemeni T. laevigatus leaves collected from Haggah [252], and the essential oil of Yemeni T. laevigatus leaves collected from Sana'a containing high amount of thymol (52.46%) [253]. The essential oil of the aerial part of Pakistani T. serpyllum (its synonym = T. laevigatus), containing carvacrol as the major component, was found to exhibit higher DPPH • scavenging activity and lipid peroxidation inhibitory effect than carvacrol [254]. Thymol was also found to scavenge DPPH • radicals and inhibit lipid peroxidation [254]. Table S1 (supplementary materials) presents more details on the mechanisms of the antioxidant activity of the studied remedies.
Wound healing can be delayed when microorganisms are present in large numbers. Therefore, reducing the bacterial load of a wound may be necessary to facilitate wound healing as well as to reduce local inflammation and tissue destruction [8]. Different extracts and isolated compounds from the studied herbal and non-herbal materials, except of tragacanth gum (Table 2), were found to possess antimicrobial activity against a wide array of microorganisms, including multi -resistant strains. The mechanisms of action for the antimicrobial activity were reported for some of the extracts and chemical constituents of these remedies. The antimicrobial activity of A. vera leaves constituents was ascribed to a number of mechanisms such as the inhibition of the microorganisms enzyme, penicillinase, by rhein, emodin and aloe-emodin [267], the inhibitory effect of aloe emodin on the initial adhesion and proliferation stages of Staphylococcus aureus biofilm development [63] and the partial disruption of virus envelopes of herpes simplex virus type 1 and type 2, varicella-zoster virus, pseudorabies virus, and influenza virus [62]. In addition, aloe emodin antibacterial and antiviral effects was attributed to the inhibition of nucleic acid biosynthesis after which protein synthesis is also inhibited [31]. Lectins and fractions of A. vera gel were reported to produce a direct inhibition of the cytomegalo virus proliferation in cell culture, perhaps by interfering with protein synthesis [20]. On the other hand, acemannan, an active constituent of A. vera gel, was found to possess indirect antimicrobial activity through its ability to stimulate phagocytic leukocytes [56]. C. recutita flowers essential oil was demonstrated to possess a virucidal activity against herps viruses by interfering with virion envelope structures or by interrupting their adsorption [110,111]. Enhancement of the susceptibility of S. aureus to a number of antibiotics (ciprofloxacin, clindamycin, erythromycin, gentamicin, tetracycline, and vancomycin) by C. recutita flowers constituents (nerolidol, farnesol and bisabolol) was attributed to the disrupting of the normal barrier function of the bacterial cell membrane, allowing the permeation of the antibiotics. This effect is more pronounced for Gram-positive bacteria, probably due to the lack of additional permeability barriers, particularly the outer membrane of Gram-negative bacteria [112]. The antibacterial properties of honey was connected to a complex interplay of various components of honey, namely, its high osmolarity, acidity, presence of bacteriostatic and bactericidal factors (hydrogen peroxide, nitric oxide, antioxidants, lysozyme, polyphenols, phenolic acids, flavonoids, methylglyoxal, bee defensin-1, and bee peptides) [130,131,136,137]. Moreover, controlling wounds malodor by honey was attributed to its antimicrobial action against odor producing bacteria and supplying copious quantity of glucose as substrate, which bacteria metabolise in preference to amino acids, from decomposed serum and tissue proteins, which are converted to malodorous substances ammonia, amines, and sulfur compounds [130,131]. Linoleic acid, a constituent of L. usitatissimum seeds was found to inhibit the growth of S. aureus by increasing the permeability of bacterial membrane due to its surfactant action [268]. M. communis extract and essential oil were reported to affect mainly the permeability of bacterial cell wall and cell membrane leading to the release of intracellular contents outside of cell and this can be accompanied with the disruption in the membrane functions such as electron transfer, enzyme activity or nutrient absorption [174]. The antifungal (against a number of Candida species (C. krusei, C. guilliermondii, C. parapsilosis, C. lusitaniae and C. rugosa)) and the antiviral activities of P. granatum L. fruit peel extract were shown to be due to morphological alterations , cell aggregation and growth inhibition of fungi [229] and impeding viral attachment and penetration via disruption of the glycosylation of viral glycoproteins [219]. The antimicrobial mechanism of action of the leaves of T. laevigatus Vahl. has not been yet illustrated. However, the antimicrobial mechanisms of action of the compounds ,carvacrol and thymol, which are also the major constituents of essential oils of Yemeni T. laevigatus Vahl leaves, growing in different areas, [252,253] were demonstrated by their ability to permeabilize, depolarize, and disrupt the cytoplasmic membrane, and inhibitory effect on biofilm formation (inhibiting the growth of preformed biofilm and interfering with biofilm formation during planktonic growth) as well as by their antifungal activity (changing cell membrane fluidiy and permeability, and changing the morphogenesis of the envelope of C. albicans) [259].
Wound healing is a dynamic and complex process involving a series of co-ordinated 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 and connective tissue and collagen deposition and finally, increasing the wound strength that takes place in an ordered manner and culminates in the repair of severed tissues [12]. Nine of the herbal and non-herbal remedies were found to promote wound healing ( Table 2). These remedies were reported to speed wound healing by several mechanisms of action including stimulating a variety of specialized cells, such as macrophages, fibroblasts, and epithelial cells and the action of cytokines, chemokines and growth factors that regulate the cellular functions during different wound healing phases as well as activating the cellular immune system. Regarding the wound healing activity of A.vera gel, there are many contradictions. Some studies, for example, showed that Aloe vera promoted the rates of healing, while, in contrast, other studies showed that wounds to which A. vera gel was applied were significantly slower to heal than those treated with conventional medical preparations [31,38,69,82]. Searching literature data has indicated that A. vera gel exerts its wound healing activity by several mechanisms of action such as keeping the wound moist, insulating and protecting, reducing inflammation, increasing epithelial cell migration, promoting collagen formation and maturation, enhancing fibroblast proliferation, direct stimulating the activity of macrophages and fibroblasts, and thus increasing collagen and proteoglycan synthesis, increasing the synthesis of glycosaminoglycans of the matrix, acting as an inhibitor of thromboxane A2 (a mediator of progressive tissue damage), promoting cell growth and attachment, increasing epithelialization and angiogenesis, accelerating wound contraction and wound closure as well as increasing oxygen access as a result of increased blood supply and stimulating the complement linked to polysaccharides [26,41,[67][68][69][70][71]73,74]. The aqueous extract of A. vera leaves (without the rind) was shown to accelerate epithelialization, wound contraction, tissue alignment and tissue strength at the later stage of wound healing [77]. The following A. vera gel active constituents were found to promote wound healing by a number of mechanisms of action: aloe emodin increased the rate of wound healing, reduced the wound area, stimulated re-epithelialization and promoted angiogenesis as a result of the stimulation of vascular endothelial growth factor (VEGF) due to an increase in the expression of IL-1β in macrophages [78], acemannan acted as macrophage activator, and stimulated fibroblast proliferation and granulation tissue formation [20,68,69], glucomannan activated macrophages and both glucomannan and acemannan stimulated immune system and possessed antibacterial and antiviral activities [20], aloeride activated transcription factor NF-kappa B and macrophages [80], mannose 6-phosphate (via binding to the insulin like growth factor receptors) stimulated the fibroblast to increase collagen and proteoglycans production and hence to increase wound tensile strength [26], mannose rich polysaccharides with molecular weight between 50 and 250 kDa regulated matrix metallopeptidase (MMP-3) and the metallopeptidase inhibitor-2 gene expression during the dermal wound repair that might also influence the granulation tissue formation and wound closure by increased production of extracellular matrix constituents including glycosaminoglycans and collagen [82], glycoprotein fraction with a molecular weight of about 5.5 kDa enhanced keratinocyte multiplication and migration, expression of proliferation of related factors, and epidermis formation [79]. In addition to the above mentioned anti-inflammatory activity of both veracylglucan B (Vglc B) and veracylglucan C (Vglc C), Vglc C was found to exhibit a significant cell proliferative effect at 100µg/mL (slightly superior to that of human plateletsderived growth factor (PDGF) at the same concentrations), while Vglc B showed a significant cell anti-proliferative effect at 1mg/mL. A remarkable antagonism of the proliferative effect of Vglc C through Vglc B was also demonstrated in this study [38]. To understand some of the reasons behind many Alasbahi and Groot contradictions about the therapeutic properties of A. vera gel, and by considering that wound healing involves cell proliferation and that a recombinant PDGF (Becaplermin ® ) is available as a remedy for poor wound-healing by diabetic ulcers, the authors concluded that Vglc C is the compound in Aloe vera gel, which is responsible for the wound healing properties and the antiproliferative activity of Vglc B is, on the other hand, responsible for the retardation and cancellation of significant healing effects arising from Vglc C, and depending on the variations in the concentration of these bioactive maloyl glucans and contamination with anthranoids (e.g., aloin) as a result of the cultivation in different climatic zones, harvesting and processing (stability problems) of Aloe barbadensis Miller to Aloe vera gel, the gel containing higher amount of Vglc B or anthranoids will lead to retardation of wound healing, while higher quantities of Vglc C would definitely foster wound healing [38]. Further constituents of A. vera such as ascorbic acid and gibberellin were found to enhance the synthesis of collagen and counterbalance collagen breakdown [41], and to stimulate fibroblasts activity and proliferation [20], respectively. The wound healing activity of C. recutita flowers extracts was attributed to their ability to produce wound drying and speed re-epithelialization after dermabrasion [68], accelerate burn wounds cleansing and improve granulation [88], exhibit marked dryness of wound margins with tissue regeneration and reduce wound area [116] and increase wound contraction, together with the increased wound-breaking strength, and hydroxyproline content [90]. Chamomile active constituents such as (-)-α-bisabolol and chamazulene were reported to shorten the healing time of cutaneous burns of guinea pigs in which (-)-α-bisabolol caused a stronger blood ciruculation. In addition, both the (-)-α-bisabolol and farnesene promoted epithelization and granulation [92]. Hydrocolloidal membrane containing cuttlefish bone (CB) was found effective in healing rats wounds by significant improving in scar tissue reduction, epithelium regeneration, angiogenesis, and extracellular matrix deposition in wound area [128]. Moreover, CB ointment (CB extract: white petroleum = 4:6)) was shown to exhibit wound healing activity of thermal burn wounds in rats by reducing the levels of white blood cells, and the expression of TNF-α, and IL-6 at late time, as well as by inhibition of lipid peroxidation and promoting re-epithelialization. These effects are comparable to those of silver sulfadiazine [122]. Testing CB extract by the same authors has revealed that treating murine macrophage cell line (RAW 264.7 cells) with CB extract induced the activation of macrophages and increased the production of pro-inflammatory cytokins such as TNF-α, IL-1β, and IL-6 as well as transforming growth factor-beta (TGF-β), VEGF and NO. On the other hand, it has been found that CB extract suppressed the production of TNF-α, IL-1β, and IL-6 cytokines in macrophages activated with LPS suggesting that CB may protect the cell and tissue from injury or destruction at high concentration of cytokines. In addition, CB extract was found to enhance proliferation of murine fibroblast and induce the activation of fibroblast to increase the expression of the matrix metalloproteases MMP1 gene and the secretion of MMP1 protein in fibroblasts, which may play an important role in the regulation of acute inflammatory reaction in a pathologic status such as burn. Furthermore, CB extract induced the production of IL-8 in macrophages, which is related to the cell migration, and treatment with CB was found to enhance fibroblast migration and invasion [123,127]. Chitin was characterized as the main component of cuttlebone by using FT-IR [122], and HPLC [127] methods. Applying scanning electron microscope indicated almost no difference in morphology between CB extract and chitin [123]. It has been concluded that the proposed mechanism of CB extract to promote healing of burned lesion of rats was associated with that chitin in CB extract, which stimulated wound skins to induce acute inflammation and promoted cell proliferation and MMP expression in fibroblast [127]. In addition, it was reported that chitin activated macrophages by interacting with cell surface receptors such as mannose receptor and toll-like receptor-2 and chitin-activated macrophages enhanced the formation of tissue in the wound by migrating inflammatory cells and the production of endothelial growth factor. Furthermore, chitin is known to play an essential role in homeostasis [123,127]. The wound healing activity of honey has been attributed to several mechanisms such as its ability to create a moist environment and consequently provide a constant flow of nutrients, help with oxygenation, supply glucose to the epithelial cells, give a constantly replenished supply of proteases to facilitate the rapid debridement of wounds, and activate proteases during debridement, promote the formation of granulation tissue, and wound epithelialization, stimulate angiogenesis, tissue growth and the synthesis of collagen, increase wound contraction, improve of the strength (cross-linking) of collagen, and tensile strength of the wounds. Moreover, it has been demonstrated that honey may work also by stimulating the activity of the immune system, e.g. by promoting the proliferation of peripheral blood B-and T-lymphocytes in cell culture, activating phagocytes from blood, stimulating monocytes in cell culture to release the cytokines TNF-α, IL-1 and IL-6, and augmenting the immune response by supplying glucose that is essential for the 'respiratory burst' in macrophages, as well as providing substrates for glycolysis, the major mechanism of energy production in the macrophages [130,131]. L. usitatissimum seeds oil has been shown to exhibit wound healing by several mechanisms of action including cicatrizing and emollient effects, less inflammatory cells in the period of re-epithelization, shortening the inflammatory stage, completing epithelium regeneration, discreet fibrosis, enhancing neo-vascularization, increasing number of collagen fibers, fibroblasts and many myofibroblasts, improving migration of fibroblasts, higher wounds contraction and shortening the healing period [117,141,146,148,149]. A topical administration of a semisolid formulation of linseed oil (SSFLO) (1% or 5%) composed of commercial linseed oil in petroleum jelly was found to promote a significant (P < .05) complete re-epithelialization of skin wounds in 100% of the animals treated compared to negative control (petroleum jelly) at the end of the experiment (14 days), but the same was not observed in groups treated with 10% SSFLO and linseed oil. The authors explained this phenomenon by the greater dermal absorption of ω-3 polyunsaturated fatty acid that presents in higher concentration in the10% SSFLO and linseed oil than in 1 or 5% SSFLO and its effect on delaying wound healing. Moreover, a significant (P < .05) amount of inflammatory cells in scar tissue was observed in the group treated with 10% SSFLO compared to negative control (petroleum jelly) at the end of the experiment (14 days) [147]. It has been demonstrated that M. communis leaves methanol extract and 10% methanol extract cream (70% methanol in paraffin oil) accelerated the healing of second-degree burn wounds in rats, comparing to sliver sulfadiazine 1%, by increasing the revascularization and fibroblast cell proliferation [186], and promoted the healing of wounds in rats by increasing wound closure, hair follicle and blood vessel numbers, skin thickness and collagen fibers [269], respectively. In addition, 80% ethanol extract (1.5 µg/mL) of M. communis leaves was found to increase the protein expression of angiogenic markers (hypoxia-inducible factor-1α (HIF-1α) and VEGF in human umbilical vein endothelial cells [156]. The wound healing activity of potassium alum was reported to be due to its adstringent property that causes contraction of tissues, constriction of blood vessels, extraction of water from tissue and precipitation of proteins, which leads to decreased capillary permeability, hardening of the capillary endothelium and reduction in oedema, inflammation and exudates [192,270]. Different extracts of P. granatum L. fruit peel such as aqueous extracts (10% and 20%) [240], 70% ethanol extract [234], a 5% (w/w) 75% methanol extract based-ointment [210] and a 10% and 15% (w/w) methanol extract based-ointment [241] were shown to enhance the healing of burn wounds and wounds by increasing wound contraction, reducing the period of epithelization and enhancing epithelialization. In addition, 70% ethanol extract was found to increase mean wound contraction (97 %) of deep second-degree skin burns in rats comparing to silver sulfadiazine (79 %) and its effect was comparable to the silver sulfadiazine in improving various phases of wound healing (reduction of inflammatory cells, increase in the formation of fibroblast, granulation tissues, collagen fibers, epithelialization and angiogenesis) [233]. Moreover, ethanol-water (3:1) extract showed wound healing percentage of 94.83% ± 0.44 comparing to the reference phenytoin with 96.00% ± 0.29 of rat's wounds after 14 days. Its effect on reducing the number of immune cells and accelerating the second stage of the healing (increasing epithelialization, neovascularization, fibroblast proliferation), and the migration of fibroblast to the wounded tissue is comparable with phenytoin [242]. Tragacanth gum gel 5%, applied topically was found to heal rats skin wounds by significant increasing of wound closure and by its high wound healing index that incorporates two factors of granulation tissue formation and epithelial regeneration [83]. Healing of skin wounds in rabbit by a 6% tragacanth mucilage (in eucerin base), topically applied, was attributed to a significant shortening of the period of wound healing and closure, which is suggested to be due to an acceleration of collagenation and proliferation phases of the wound repair [84].
Various phytochemical constituents, isolated from the studied herbal and non-herbal remedies, were found to contribute in wound healing activity via one or more of their pharmacological activities (anti-inflammatory, antioxidants, and antimicrobial activities as well as the promotion of one or several phases of wound healing process) ( Table 2). These bioactive constituents belong to carbohydrates (monosaccharides (mannose-6-phosphate), disaccharide (veracylglucan B), oligosaccharide (veracylglucan C) and the polysaccharides (acemannan, glucomannan, aloeride, mannose rich polysaccharides and several other polysaccharides designated as APS-1, APS, GAPS-1 and SAPS-1, obtained from Aloe vera, and the polysaccharides of cuttlefish bone and chitin as the main constituent of cuttlefish bone), phenolic compounds such as simple phenol (pyrocatechol), phenolic acids (salicylic acid, p-coumaric acid, gallic acid, ellagic acid), polyphenolic compounds (myrtucommulone, semimyrtucommulone, gallo-myrtucommulone A & B), flavonoids (apigenin, apigenin-7-O-(6"-acetyl)-glucoside, apigenin-7-glucoside, chrysosplenol D, eupatolitin, luteolin, myricetin, myricetin derivatives, quercetin, rutin, herbacetin 3,7-O-dimethyl ether and its aglycone herbacetin, gallocatechin and its derivatives, catechin gallocatechin), chromones propyl-7-methoxy-5-methylchromone, isorabaichromone, aloesin, aloeresin A, aloeresin B), coumarins (dihydrocoumarin and dihydrocoumarin ethyl ester, umbelliferone), anthraquinones (aloin, emodin, aloe emodin), hydrolyzable tannins such as ellagitannins (oenothein B, eugeniflorin D2, tellimagrandins I and II, punicalagin, punicalin, pedunculagin, strictinin A, granatins A and B, casuarinin, corilagin, gallagyldilacton), caroboxylic acid (cinnamic acid), tetrahydroxy-cyclohexane carboxylic acid (quinic acid 3,5-di-O-gallate), terpenoids (monoterpenoids (carvacrol, thymol), sesquiterpenoids (α-bisabolol, (-)-α-bisabololoxides A and B, chamazulene, chamaviolin, en-yn-dicycloethers, farnesene, farnesol, matricin, nerolidol), fatty acids (α-linolenic acid, linoleic acid, oleic acid, palmitoleic acid, palmitic acid, and stearic acid), saponins, sterols, proteins, peptides, hormones, enzymes, and vitamins (Table 2). Several studies have revealed the beneficial role of phytochemicals in wound healing. The role of various polysaccharides in the healing of wounds and burns, via their immunomodulatory effects, antioxidant properties, macrophage activation, controlling the inflammatory responses, stimulating wound contraction, accelerating the phases of re-epithelialization and remodelling, has been demonstrated in a number of studies [118,[271][272][273][274]. Phenolics derived from various natural sources are linked to antioxidant, antiinflammatory, antiallergic, anticarcinogenic, antihypertensive, cardioprotective, anti-arthritic and antimicrobial activities. The antioxidant activity of phenolics is primarily attributed to their redox properties that enable them to act as singlet oxygen quenchers, reducing agents and their hydroxyl (-OH) groups are good H-donating antioxidant agents that disrupt the cycle of new radical generation by scavenging reactive oxygen species. Various studies validated the positive correlation between phenolic content and the antioxidant activity [275]. Positive correlation between the antioxidant activity and phenolic content was also observed by the extracts of A. vera leaves [22,47], C. recutita flowers [100,102], M. communis leaves [162,163,184], and P. granatum peel [201,203,212,213,215,216,276]. For R. nervosus leaves, a correlation was observed between the principal polyphenolic components (flavonols) of the extract and the antioxidant activity [244] (Table 2; Supplementary material, Table S1). The inhibitory effects of polyphenolic compounds against bacterial Alasbahi and Groot pathogens have been ascribed to their ability to attack several targets of pathogenic microorganisms such as the inhibition of nucleic acid synthesis, the perturbation of the cytoplasmatic membrane and thereby affecting the permeability and leading to intracellular constituent release and the inhibition of energetic metabolism and thus interfering with membrane functions such as electron transport, nutrient absorption, nucleic acid synthesis, and ATPase activity [277]. Although the precise mechanisms of the anti-inflammatory activity of phenolic compounds are not fully elucidated, it is has been hypothesized that phenolic compounds exert anti-inflammatory activity by inhibition the synthesis of pro-inflammatory mediators, modification of eicosanoid synthesis, inhibition of activated immune cells, or inhibition of nitric oxide synthase and COX-2 via the inhibitory effects on nuclear factor NF-κβ [278]. Several monoterpenoids including thymol were shown to accelerate wound healing via their anti-inflammtory, antioxidant, and antimicrobial activities as well as modulation of some wound healing phases [279]. In vivo and in vitro experimental studies have indicated the potential anti-inflammatory activity of sesquiterpenes via modulating or suppressing elements that play a direct role in the inflammatory response [280]. Saponins are effective in wound healing due to their antioxidant and antimicrobial activities, which appear to play a role in wound contraction and elevated rate of epithelialization [8]. Phytosterols were reported to possess antioxidant and antimicrobial activities [281][282][283][284]. The role of vitamins in accelereating wound healing due to their antioxidant, anti-inflammatory and immunomodulatory activities as well as their stimulatory effects on various phases of tissue healing has been evaluated in several studies [285,286].
The knowledge of the side effects and/or risks associated with the use of herbal and non-herbal remedies is very crucial in order to promote awareness among herbalists, health professionals and the public on the risks associated with excessive or chronic use of herbs. Aloe vera gel topical application has been reported to cause a number of side effets such as contact and photodermatitis and/or erythema with papulous, acute skin rash, burning sensation in some patients, and mild itching. All adverse effects were reversible and Aloe vera was generally well tolerated [28,70]. Chamomile is considered safe to use topically and orally and is included in the FDA (Food and Drug Administration, USA) GRAS (generally recognized as safe) list [287,288]. However, both oral and topical uses of chamomile flowers have been reported to cause contact dermatitis, particularly among those who also have allergies to other plants in the daisy family (Asteraceae or Compositae) [89,287,288]. The tragacanth gum is also considered as GRAS and approved as a food ingredient (emulsifier, stabilizer, thickener and gelling agent) by the FDA, and by the European Union and has been accorded with E413, a European food safety E number [289]. Honey poses a small risk of wound infection as it may contain some clostridial spores. However, this risk can be reduced by using honey treated with gamma-irradiation, which can kill the spores while maintaining honey's antibacterial activity. On the other hand, there has not been a single occurrence of wound infection contributed by clostridial spores with the topical application of honey in approximately 2000 cases reported in 2014. Although there may be some toxic effects from the ingestion of poorly handled honey, there have not been any documented toxic effects associated with the topical application of honey on diabetic wounds in comparison with the risk of using other conventional wound healing therapies. Besides these few limitations, many studies reported honey as a non-toxic, non-allergenic, non-irritating healing agent with no cytotoxic effects; it is a safe, cheap, and effective healing agent [131]. Potassium alum is considered by the FDA as GRAS substance. It is used in different products like food or drugs as buffer, neutralizing or forming agent [290]. Furthermore, we did not find any report on possible side effects following the topical use of cuttlefish bone, L. usitatissimum seeds oil, M. bengalensis leaves, M. communis leaves, P. granatum pericarp, R. nervosus leaves, and T. laevigatus leaves. Consequently, the herbal and non-herbal remedies used in Sana'a for the treatment of wounds and burn can be considered generally safe upon a proper usage in quantity and manner.

CONCLUSION
This work provides scientific data on the pharmacological activities (anti-inflammatory, antioxidant, antimicrobial and wound healing activities) of twelve herbal and non-herbal remedies that could justify the claimed usefulness of these remedies for their traditional use in Sana'a for the treatment of wounds and burns. This study opens the opportunities for further evaluation of the effectiveness and safety of the remedy's raw materials and determination of the rational way of their using either as a single use and/or in combinations. Moreover, further research of the raw materials and their associated active compounds is needed to develop useful alternatives for wound healing.
Alasbahi and Groot