PHARMACOLOGY:

1) Antiurolithiatic activity:

Methanolic bark extract of Lawsonia inermis demonstrated antiurolithiatic activity, was evaluated by performing ethylene glycol induced urolithiasis model, at doses of 300 and 500 mg/kg, p.o., respectively¹⁵.

An aqueous, ethanol leaf extract of Lawsonia inermis demonstrated antiurolithiatic activity, was evaluated by performing ethylene glycol with ammonium chloride model, at doses of 100, 200 and 400 mg/kg, p.o., respectively¹⁶.

2) Memory enhancing activity:

The petroleum ether leaf extract was found to enhance memory, reduce anxiety and affect behaviour by modifying serotonin- and norepinephrine-mediated behaviour. In addition, the extract enhanced clonidine-induced hypothermia and lowered lithium- induced head twitches in rats¹⁷.

3) Hepatoprotective activity:

The hepatoprotective effects of henna extracts are associated with the polar extracts and therefore most likely with the presence of phenolic compounds, corresponding to the ethnopharmacological use of decoctions for the treatment of jaundice and other liver disorders in India and Pakistan^18,19,20. Polar extracts were demonstrated to have a protective effect against CCl₄-induced liver toxicity in mice and rats^21-23. A dose of 400 mg/kg, p.o. administered to mice afforded protection by significantly decreasing the levels of bilirubin²⁴, while total serum proteins and bilirubin were decreased in rats by the aqueous extract at 250, 500 and 750 mg/kg. Ethanol and aqueous bark extracts were also found to inhibit CCl₄-induced oxidative stress and reduce hepatic damage in rats. The levels of serum transaminases and LDH were decreased at doses of 250 and 500 mg/kg ²⁵. A polyherbal formulation containing aqueous extract of henna, sold under the name Hepjaun syrup, was found to decrease hepatic damage in rats, consistent with decrease in bilirubin at an oral dose of 500 mg/kg, b.w.²⁶.

4) Anticancer activity:

An aqueous leaf extract of Lawsonia inermis possesses strong antitumor activity in both DMBA-induced 2-stage skin carcinogenesis and B16F10 melanoma tumour models in mice at a dose of 1000 mg/kg, p.o. The extract caused a significant decrease in the number of papilloma compared to the negative control, as well a reduction in the tumour incidence (66%), when compared to 100% tumour incidence in the control group. Moreover, the average number of papillomas (tumour yield) per mouse was found to be 1.6, compared to that of the DMBA-treated control (3.5)²⁷.

An aqueous extract from the whole plant was found to be active against Ehrlich ascites carcinoma cells and significantly decreased the number of cancer cells when applied at a dose of 10 mg/kg/day in animal models²⁸. The oral administration of aqueous leaf extract (0.3 % w/v) produced tumour suppress or effects and prolonged the mean survival and average survival time in mice with gluteal sarcoma formed with Ehrlich ascites tumour cells²⁹.

A study indicated promising cytotoxicity of a chloroform extract from henna against human colon cancer (Caco-2) and liver cancer (HepG2) cell lines, with corresponding IC₅₀ values of 25.1 and 28 μg/mL, respectively³⁰. The photo cytotoxicity of methanol leaf extract against a human leukaemia cell-line HL60. Cancer cell viability reduced by more than 50% following exposure to an effective concentration of 20 μg/mL under broad spectrum light (9.6 J/cm²)³¹. Ethanol leaf extract of henna displayed significant (p<0.001) anticarcinogenic activities against benzo(a)pyrene- induced forestomach and 7,12-dimethylbenz(a) anthracene- initiated croton oil promoted skin papilloma genesis models at doses of 200 and 400 mg/kg, p.o. respectively, by reducing the tumour burden and tumour incidence in mice³². Ethanol root extract displayed antitumor activity against Dalton's lymphoma ascites-induced mice at a dose of 180 mg/kg, p.o. by reversing the increases in WBCs, platelets and lymphocytes, and also by decreasing RBCs, haemoglobin content and monocytes³³. Chloroform leaf extracts were cytotoxic towards human colon carcinoma Colo-205 cancer cells by 59% proliferation at IC₅₀ 86.77 μg/mL ³⁴.

A cream prepared from a mixture of henna leaf and Plumbi oxidum was applied intravaginally to adult women at the National Institute of Unani Medicine, Bangalore, India, to treat cervical erosion. This condition is thought to be a primary stage of cancer. It was reported that 27% of the patients were completely healed of erosion when compared to untreated patients³⁵.

Essential oil isolated from the leaves exhibited strong cytotoxic activity (IC₅₀=24 μg/mL) against liver cell lines (HepG2)³⁶. An isoplumbagin, isolated from the stem bark of henna, displays cytotoxicity towards melanoma and colon cancer cell lines, as well as against several non-small cell lung, colon, CNS and renal cell lines³⁷.

The finding that an ethanol leaf extract displayed cytotoxicity towards normal human amniotic epithelial FL-cells, as reflected by an IC₅₀ of 75 μg/mL in the neutral red uptake assay³⁸.

5) Antiarthritic activity:

An aqueous ethanol leaf extract demonstrated antiarthritic activity, as reflected by a reduction in paw oedema, paw diameter and body weight loss in both Freund's adjuvant-induced and formaldehyde-induced arthritis mice models, at doses of 200 and 400 mg/kg, p.o., respectively³⁹. In this study, an oral dose of 10 mg/kg of diclofenac sodium was used as the positive control.

6) Antiulcer activity:

An aqueous, ethanol and chloroform leaf extracts demonstrated strong anti-ulcer activity in pylorus ligation and aspirin-induced rats when compared to ranitidine, the positive control. In addition, significant reductions (p<0.001) in gastric acid secretions, total acidity and ulcer index were observed⁴⁰. An ethanol leaf extract for reducing indomethacin-induced gastric ulcers in pylorus ligation rat models by reducing the ulcer index for all three doses (100, 200 and 400 mg/kg, p.o.) tested⁴¹. The anti-ulcer activity of leaf extracts was found that aqueous, ethanol and chloroform extracts produced significant (p<0.001) activity against acute and chronic gastric ulcers in two rat models at doses of 200 and 400 mg/kg, p.o. when compared to the negative control gum acacia (2%,w/v). Sucralfate (250 mg/kg) served as the positive control. Aqueous, ethanol and chloroform extracts were found to reduce ethanol-induced ulcers by up to 81, 94 and 88%, respectively, and cold-restraint stress-induced ulcers by up to 56%, 30% and 56%, respectively⁴².

7) Antituberculostatic activity:

Aqueous leaf extract was shown to have promising antitubercular activity, both in vitro and in vivo. The extract inhibited the growth of Tubercle bacillus, from sputum, and Mycobacterium tuberculosis H 3 7Rv on Lowenstein Jensen medium at a concentration of 6 μg/ml. It was also found to combat Mycobacterium tuberculosis infection in guinea pig and mice models at a dose of 5 mg/kg, p.o.⁴³.

8) Antidiabetic activity:

Polar extracts of leaves have been investigated by several researchers to establish their antidiabetic activities. Methanol leaf extract was found to have hypoglycaemic activity in a glucose oxidase assay, both in neutral and basic media⁴⁴. Diabetic and euglycemic rats exposed to an ethanol leaf extract of henna presented with reduced levels of blood glucose at a dose of 200 mg/kg, b.w., when compared to those treated with glibenclamide⁴⁵. This result was confirmed that ethanol leaf extract caused a decrease in blood glucose, total cholesterol and triglyceride concentrations from 194 mg/dL to normal, 148.9 to 55.3 mg/dL and 225.7 to 76.9 mg/dL, respectively, in alloxan-induced diabetic mice at an oral dose of 800 mg/kg⁴⁶. A similar study found that methanol leaf extract produced antidiabetic activity in alloxan-induced diabetic rats at a dose of 600 mg/kg, while significantly reducing blood glucose, serum triglycerides and cholesterol levels by 34.33%, 43.35% and 34.18%, respectively⁴⁷. A recent report indicated that methanol leaf extracts, at a concentration of 10 μg/mL, caused a 61% inhibition of amylase activity when compared to that of acarbose (10 μg/mL)⁴⁸.

9) Antibacterial activity:

Although antibacterial studies of henna extracts have been documented as early as 1968⁴⁹, research still continues. The growth inhibition effects of the plant extract have been evaluated against an extremely large variety of human pathogenic bacteria. In most cases, aqueous extracts, employing hot or cold water, methanol, ethanol and even acetone, of the leaves or whole plant, and in a few cases, of the stems and bark, have been found to have the highest efficacies. This corresponds to the traditional intake of decoctions prepared from the leaves of henna for a variety of ailments associated with bacterial infections^{50,
51, 52, 53}. Ethyl acetate and ethanol extracts from flowers and fruit exhibited significant (p<0.05) inhibitory effects against Staphylococcus aureus, Pseudomonas aeruginosa, Escherichia coli and Bacillus subtilis at 10 mg/mL⁵⁴. Aqueous leaf extract showed in vivo antibacterial activity against Staphylococcus aureus, Pseudomonas aeruginosa and Escherichia coli with IZD values of 10, 8 and 16 mm, respectively at 10 mg/mL when the leaf segments of henna plant were used as in vivo samples⁵⁵. Only a few reports document activity observed from non-polar extracts. Aqueous, ethanol, but also chloroform and petroleum ether extracts from the whole plant, displayed good activity against Gram-positive (Bacillus cereus, Bacillus subtilis, Staphylococcus aureus) and Gram-negative (Escherichia coli, Proteus vulgaris, Pseudomonas aeruginosa) bacteria, as evaluated by the disc diffusion method. The IZD values ranged from 1.6 to 3.8 cm when compared to streptomycin (1.6–2.8 cm) and spectinomycin (2.0– 3.1cm)⁵⁶. Diffusion methods used in most studies are not regarded as appropriate to a polar compounds, since diffusion of these compounds through the polar growth medium is restricted, leading to possible under estimation of their antibacterial potential. Serial dilution methods may yield more reliable results and have been applied to henna extracts. Ethanol extract from the twigs inhibited the growth of Staphylococcus aureus, Sarcina lutea and Shigella dysentriae in a serial dilution assay⁵⁷. There searchers reported MIC values of 165,156 and 156 mg/mL, respectively, compared to that of tetracycline, used as the positive control (MIC=30 μg/mL for all three organisms).

In spite of the large number of phytochemicals isolated from henna, few studies have compared the antibacterial activities of the extracts with the activities of the pure compounds.

Gallic acid, lawsone and 1,4-naphthoquinone were found to be responsible for the inhibitory activities of aqueous and chloroform leaf extracts against Staphylococcus aureus, Staphylococcus albus, Staphylococcus citreus, Sarcina lutea, Streptococcus faecalis, Corynebacterium pyo, Corynebacterium sp., Shigella dysenteriae, Shigella flexneri, Escherichia coli, Klebsiella aerogenes, Mycobacterium phlei, Salmonella paratyphi, Bacillus subtilis and Pseudomonas aeruginosa with IZDs ranging from 15 to 26 mm at concentrations of 60–80 mg/mL^{58, 59, 60}. A comparative antimicrobial study of a naphthoquinone-rich leaf extract (500 mg/mL), synthetic lawsone (500 mg/mL) and tetracycline (50 mg/mL), and found that leaf extract inhibited the growth of Staphylococcus epidermidis with both bactericidal and bacteriostatic activities (IZDs 16 and 27mm, respectively). The positive control (tetracycline) only displayed bactericidal activity (IZD 26mm), whereas synthetic lawsone had only a bacteriostatic effect (IZD 29mm)⁶¹. A partially identified proteinaceous compound isolated from the seeds exhibited activity against Pseudomonas aeruginosa and Staphylococcus aureus at IC₅₀ values of 11.4 and 16.6 μg/mL, respectively⁶². A lack of data is evident in the literature concerning the antibacterial activities of pure compounds and combinations of pure compounds from henna. No information regarding the possible additive, synergistic or antagonistic effects of these compounds is available.

10) Analgesic and Anti-inflammatory activity:

Lawsochylin A, luteolin, apigenin, 4-hydroxy-α-tetralone, 2-butoxysuccinic acid and lawsonaphthoate A, isolated from the stems and leaves of henna, exhibited promising anti-inflammatory activities. These compounds were found to inhibit superoxide anion generation and elastase release from human neutrophils, in response to fMLP or cytochalasin-B, at IC₅₀ ranges of 0.75 to 1.90 μg/mL and 1.58 to 3.61 μg/mL, respectively⁶³. Non-volatile oil obtained from the seeds of Lawsonia inermis was reported to have analgesic activity⁶⁴.

Phenolic compounds, present in the polar extracts, have been associated with the anti-inflammatory properties of henna. However, the naphthoquinone, lawsone, does not appear to have strong anti-inflammatory activity, compared to other compounds in the plant.

Lawsone, isolated from the chloroform fraction, was found to have moderate anti-inflammatory activity at 500 mg/kg, in comparison to the positive control, phenylbutazone (100 mg/kg). In a separate study⁶⁵, the anti-inflammatory activities of isoplumbagin and Lawsaritol from a combination of roots and stems of henna were investigated. A dose of 100 mg/kg p.o. caused a 60% and 40% reduction in swelling, respectively, when compared to phenylbutazone (61% reduction at 100 mg/kg), in a carrageenan-induced paw oedema assay in rats. The role of polar compounds in the anti-inflammatory action of henna. Aqueous leaf extract was found to reduce inflammation in carrageenan-induced hind paw oedema in rats at a dose of 250 mg/kg, p.o. whereas diclofenac sodium (5mg/kg) was used as the positive control. The ethanolic leaf extract did not demonstrate any activity⁶⁶. However, A methanol leaf extract exhibited significant anti-inflammatory activity (p<0.01) in an acetic acid induced writhing test in mice. In addition, the extract affected a reduction in chemically-induced nociceptive pain stimuli, comparing favourably to the positive control, diclofenac sodium (20 mg/kg)⁶⁷.

11) Nematicidal effect:

A suppressive effect was obtained by L. inermis against Meloidogyne incognita development. Henna reduced tomato root gall numbers, number of the egg-laying females and rate of the nematode reproduction, when tomato and henna were grown together. Also, same reduction in the nematode biological processes was found, when tomato plants were grown in soil containing root exudates of henna, but with less amount. When henna was grown alone, root gall index and the rate of nematode production reduced to 75% and 99%, respectively, compared with those of tomato grown alone⁶⁸.

12) Antifungal activity:

Lawsonia inermis has been used extensively as a traditional remedy in the form of leaf powders and pastes to cure a host of skin-related disorders^69,70,71,72. It is therefore not surprising that many researchers have investigated the activities of extracts from the aerial parts against fungal pathogens of the skin. Their studies provide evidence of the antifungal activity of polar leaf extracts of henna and some examples are provided. Ethanol, ethyl acetate and hexane extracts from whole plant exhibited potent activity against two dermatophytes, Tinea rubrum (athletes foot) and Trichophyton mentagrophytes, with MIC values ranging from 31.3 to 62.5 μg/mL⁷³. Later studies^74,75 reported lower efficacies (MIC values from 1 to 10 mg/mL) form ethanol, ethanol, acetone, petroleum ether, chloroform and ethyl acetate leaf extracts against Trichophyton tonsurans, Trichophyton mentagrophytes, Tinea rubrum, Microsporum gypseum and Epidermophyton floccosum. At concentrations of 0.25–4.0 %, the chloroform, methanol and aqueous leaf extracts of henna completely inhibited fungal growth of Malassezia, an opportunistic skin pathogen, in vitro⁷⁶. Some in vivo evidence to support in vitro findings regarding the effects of henna on skin conditions. They applied henna paste to 50 calves infected with ringworm and reported that the lesions were completely healed within 30 days⁷⁷.

Candida albicans is an opportunistic yeast that may cause skin infections, but also affects the mouth, gut and vagina⁷⁸. Polar leaf extracts were found to effectively inhibit the growth of Candida albicans and Candida tropicalis with reported MIC values ranging from 0.200 to 1.25 mg/mL^79,80. Agar well diffusion assay and TLC-bioautography of an ethanol leaf extract⁸¹ was found to be effective against Candida albicans with IZD values ranges from 31 to 40 mm at 150 mg/mL when compared to the commercial antifungal drugs fluconazole (10 μg/disc). Aqueous leaf extract showed in vivo antifungal activity against Candida albicans with IZD value of 10 mm at 10 mg/mL when the leaf segments were used as in vivo samples⁵⁵. Ethanol extract of leaves of L. inermis showed significant antifungal effect against phytopathogenic fungi. Ethanol extract could be used as alternative source of antifungal agents for protection of plants or crops against fungal infection⁸².

13) Antiviral activity:

Aqueous leaf extract, at an effective concentration of 125 μg/mL, achieved a 60 % retroviral reverse transcriptase inhibition of Moloney murine leukaemia virus reverse transcriptase, reacted with 3H-dTTP⁸³. A methanol extract was also demonstrated to produce antiviral activity against mammalian Sindbis virus at a MIC of 1.5 μg/mL⁸⁴. Not much attention has been paid to the antiviral activities of henna in recent years.

14) Anthelmintic activity:

Aqueous leaf extract was shown to have anthelmintic activity against adult individuals of the tropical flatworm, Fasciola gigantica. A significant reduction in motilities and an increase in mortalities were recorded at an effective concentration of 5 % (w/v) when compared to that of oxyclozanide (5 % w/v)⁸⁵.

15)Antiparacidal activity:

Methanol and dichloromethane extracts obtained from the leaves displayed antiparacidal activity against Plasmodium falciparum (IC₅₀ > 20 μg/mL for both extracts), Trypanosoma brucei (LC ₁₀₀ 6.2 and 1.5 μg/mL, for the methanol and dichloromethane extract, respectively), Leishmania donovani (EC₅₀>100 μg/mL for both extracts) and Nematoda (EC₅₀ >10 μg/mL for both extracts). The extracts were in effective towards mites (EC₅₀>3000 mg/m² for methanol extract only). Artemether, chloroquine, melarsoprol, pentamidine, ivermectin and benzylbenzoate were used as positive controls⁸⁶.

Aqueous leaf extract was found to have an antilousicidal action against the human head louse (Pediculus humanus capitis) and the sheep body louse (Bovicola ovis) with LC₅₀ values of 18.26 and 21.19 mg/L, respectively⁸⁷.

16)Abortifacient activity:

The methanol root extract of henna produced an abortifacient effect in various animal models and attributed the effect to the toxicity of the extract towards both mother and foetus⁸⁸. An aqueous root extract was found to induce spontaneous abortion in pregnant rats over a dose range of 800–1600 mg/kg⁸⁹. An aqueous extract administered to rats exhibited a higher uterotonic activity, related to early trimester abortion, than oxytocin. Contractions of the uterus were induced in both non-pregnant and pregnant rats over the dose range of 50–800 μg/mL. A LD₅₀ dose of 894 mg/kg p.o. was recorded⁹⁰.

17)Antioxidant activity:

Many researchers have reported on the anti-oxidant activities of henna extracts. Polar extracts of leaves ^91,92,93, fruit and seeds^94,95 in particular and the leaf essential oils⁹⁶ were found to have good activities in a variety of anti-oxidant assays, when compared to positive controls. The anti-oxidant activities of some of the pure compounds, including lawsone, confirmed the role of the phenolic compounds in the observed activities. The methanol extract from leaves for anti-oxidant activity using FTC and TBA assays and for free radical scavenging activity using the DPPH assay. The extract at 0.02% displayed a significant activity in both FTC and TBA assays, when compared to that of the negative controls. Furthermore, the same extract caused a 67.7% decolourization of DPPH compared to the positive controls, α-tocopherol (49.3%) and BHT (58.16%)⁹¹. The methanol leaf extract from henna, collected from the Dead Sea region, indicated good activity as reflected by a chlorogenic acid equivalent of 71.5 mg/g (dry weight) against DPPH radicals. A high correlation was also established between the anti-oxidant activity and the total phenolic contents (gallic acid equivalent ¼ 39.2 mg/g dry weight) of the plant, indicating that the phenolic compounds may be responsible for the activity⁹². Ethanol and aqueous leaf extracts were found to have good DPPH scavenging activities (IC₅₀ values of 1.3 and 3.7 mg/mL, respectively), when compared to that of positive controls, grape seed (0.46 mg/mL), green tea (0.28 mg/mL), emblica (0.31 mg/mL) and vitamin C (0.01 mg/mL)⁹⁷. The same group⁹⁸ compared the anti-oxidant activities of leaf extracts against DPPH, galvinoxyl and ABTS and reported IC₅₀ values 1.3, 0.32 and 0.11 mg/mL, respectively, for the ethanol extract and 3.70, 1.20 and 0.93 mg/mL, respectively, for the aqueous extract. Grape seed extract was used as positive control in this study, with IC₅₀ values of 0.27, 0.09 and 0.04 mg/mL for the ethanol extract and 0.46, 0.48 and 0.19 mg/mL for the aqueous extract, as evaluated using the DPPH, galvinoxyl and ABTS assays, respectively. An ethanol and aqueous extracts from the whole plant are able to inhibit lipid peroxidation and demonstrate cytoprotective action against Cr (VI)-induced oxidative cellular damage⁹⁹. An oral dose of aqueous leaf extract at 1000 mg/kg induced anti-oxidant enzyme activity in serum, kidney and liver cells by increasing the lipid peroxidase and hydrogen peroxidase levels in rats when compared to that of the positive control ascorbic acid (180 mg/kg)¹⁰⁰. An anti-oxidant activity, measured in a DPPH assay, of methanol leaf extract, gained through a controlled microwave-assisted hydro thermal process at 100–120 1C, was due to the presence of phenolic compounds¹⁰¹. An n-butanol leaf extract was found to prevent the oxidation of linoleic acid¹⁰². Phenolic glycosides isolated from the extract displayed strong antioxidant activity against DPPH and the most active compound was identified as 1, 2, 4-trihydroxynaphthalene-1-O-β-D-glucopyranoside (EC value 50 μg/mL). Leaf essential oil, tested at a concentration of 0.02 %, was found to have similar antioxidant activities in the FTC and TBA assays as α-tocopherol⁹⁶. The high anti-oxidant efficacy of henna fruits, as established by a DPPH assay, was attributed to the presence of phenolic compounds⁹⁴. An ethanol extract obtained from the seeds displayed better anti-oxidant activity than ascorbic acid in a DPPH assay. The extract was not only able to reduce Mo (VI) to Mo (V) and Fe (III) to Fe (II), but also inhibited lipid peroxidation⁹⁵. Lawsone, apigenin, luteolin, cosmosiin, p-coumaric acid, 2-meth- oxy-3-methyl-1, 4-naphthoquinone and apiin, produced by the leaves, displayed good antioxidant activities against ABTS when compared to ascorbic acid¹⁰³.

18) Protein glycation inhibitory activity:

An ethanol extract from the plant caused inhibition of protein glycation and reduced protein damage induced by a free radical generator¹⁰⁴. This extract, lawsone and gallic acid from the plant inhibited the formation of advanced glycated end-products by 77.95 %, 79.10 % and 66.98 % at concentrations of 1500 μg/mL, 1000 μg/mL and 1000 μM, respectively. In addition, glycation was inhibited with corresponding IC₅₀ values of 82.06 μg/mL, 67.42 μM and 401.7 μM, respectively.

19) Wound healing activity:

An ethanol leaf extract promoted wound healing by 97%, compared to the control, in both excision and incision wound models in rats. Flavonoids, including luteolin, were associated with the activity. The petroleum ether extract was also effective, but only in incision wound models. Lawsone from the leaf extract was found to promote healing of both excision and incision wounds in rats, when administered orally. However, the leaf extract, as well as lawsone, were found to be more effective wound-healing agents when applied externally in the form of an ointment^105,106. The wound healing activity of ethanol leaf extract was evaluated on excision, incision and dead space wound models of rats. The extract at a dose of 200 mg/kg showed 71% reduction in the wound area on all models when compared with control (58%). Rapid wound contraction and high skin breaking strength was observed in treated rats, com- pared to those treated with nitrofurazone ointment, the control^107,108.

20) Molluscicidal activity:

Leaf, bark and seed of henna against Lymnaea acuminata and Indoplanorbis exustus were studied. Seed powder was more toxic than leaf and bark against I. exustus. Binary combinations of henna seed with Cedrus deodara Roxh and Azadirachta indica A. Juss oil, or powdered Allium sativum, or Zingiber officinale rhizome oleoresin was more toxic to snails L. acuminata and I. exustus than their single treatment. The highest increase in the toxicity was observed when henna seeds powder and C. deodara oil (1:1) were tested against both the snails. The combination with neem oil was also more toxic than their individual components and other combinations¹⁰⁹.

21) Antitrypanasomal activity:

Ethanolic leaf extract and pure lawsone were found to reduce the availability of biologically active trypsin, an enzyme essential to human and animal nutrition. Trypsin inhibition was achieved in vitro at IC₅₀ values of 64.87 and 48.6 μg/mL, respectively¹¹⁰. More than 50 % reduction in tyrosinase activity was achieved by a methanol extract of henna at a concentration of 1140 mg/L¹¹¹, whereas 2-(β-D-glucopyranosyloxy)-1, 4-naphthoquinone and 7- hydroxy-4-methylcoumarin isolated from the stems inhibited the urease activity by more than 50 % at a concentration of 500 μM¹¹².

22) Antifertility activity:

Administering of henna leaf powder from the plant, known as avrodhak, at daily doses of 3–300 mg/kg p.o., reduced the pregnancy rate of rats by 40–60 % ¹¹³. The effect was permanent, indicating that henna promotes infertility in rats. An ethanol leaf extract discouraged implantation in a female Wistar rat model at a dose of 134 mg/kg p.o.¹¹⁴. Evidence that henna extracts reduce fertility was provided by¹¹⁵ using healthy female and male Wistar rats, dosed with henna root extract. In addition to a loss in body weight, the number of implementation sites reduced in a dose-response fashion. A significant reduction in the number of corpora lutea was measured in all experimental and positive control (Ethinyl estradiol) groups. The root extracts were positive in tests for steroids, suggesting that phytosterols may be responsible for the antifertility effects of the extract.

23) Ovicidal activity:

The Ovicidal activity of henna was demonstrated by exposing eggs of the bean weevil (Callosobruchus chinensis) to acetone and petroleum ether extracts from the leaves. A 85% reduction in egg hatchability was observed in acetone extract whereas a 71% reduction was recorded for petroleum ether extract at the highest concentration of 100 % (w/v) for both extracts¹¹⁶. In a separate study, eggs of the diamond back moth (Plutella xylostella) were exposed to acetone leaf extract at concentrations ranging from 25 % to 100 % (w/v).The maximum concentration (100%) increased egg mortality by 62.5 % ¹¹⁷, but the eggs were not affected at the other concentrations.

24) Antidermatophytic activity:

The antidermatophytic activity of ethanol, ethyl acetate and hexane extracts of L. inermis were tested on 5 strains each of Tinea rubrum and Tinea mentagrophytes. All these extracts showed significant antidermatophytic properties in-vitro¹¹⁸.

25) Anticonvulsant activity:

Anticonvulsant activity of Hydroalcoholic extract of Lawsonia inermis (HAELI) leaves was evaluated against Pentylenetetrazol (PTZ) and Isoniazid (INH) induced convulsions in Swiss Albino Mice at the dose of 200 and 400 mg/kg and HAELI shows anticonvulsant activity in both PTZ and INH induced convulsion in mice¹¹⁹.

26) Immunomodulatory activity:

Henna leaf extract containing naphthoquinones stimulated human neutrophils in vitro at dose ranges of 1–7 μg/mL. The same extract stimulated macrophage phagocytic activity in mice at 5 mg/kg¹²⁰.

Methanolic extract of Lawsonia inermis leaves at 1 mg/ml concentration had displayed immunostimulant action as indicated by promotion of T-lymphocyte proliferative responses, fractionation of the total methanolic extract of henna leaves. Naphthoquinone fraction obtained from leaves L. inermis showed significant immunomodulatory effect^121,122.

27) Nootropic activity:

The acetone fraction of the petroleum ether extract of Henna inhibited prominent nootropic activity, potentiating clonidine induced hypodermia and decreased lithium induced head twitch. The fraction modified 5-HT and NA mediated behaviour. However haloperidol induced catalepsy was not modified¹²³.

28) Thrombolytic activity:

An aqueous leaf extract displayed excellent thrombolytic activity, as reflected by a 62.4 % weight reduction of clots formed in vitro, when compared to the same amount of streptokinase, which resulted in an 84.6 % weight loss¹²⁴.

29) Anticataleptic activity:

Significant action was obtained by treating haloperidol-induced catalepsy in mice with an aqueous extract of henna. A reduction in cataleptic scores was found and an increase in superoxide dismutase activity was measured at a dose of 400 mg/kg¹²⁵.

30) Antisickling activity:

Aqueous extract of leaves of L. inermis was found to inhibit sickling and to increase the oxygen affinity of HbSS blood¹²⁶.

31) Miscellaneous activity:

A traditional Unani formulation comprising henna, Olea europea and Nigella sativa was reported to soothe eczema by reducing itching, burning, erythema and oedema by upto 80 % after 42 days of treatment in human patients¹²⁷. Henna applied as a dye was found to be active against so-called hand-foot syndrome, a common side effect of synthetic medicines, including the cancer treatment drug, Capecitabine^128,129. Henna pigment was found to be a carrier of antigens following transcutaneous vaccination of mice. However, it did not produce any detectable antigenic response when suspended with crude antigen of Paracoccidioides brasiliensis or bovine serum albumin. No differences were found in the production of NO, interleukin-4 and γ-interferon in the supernatants of the corresponding cell cultures¹³⁰. A petroleum ether extract of henna reportedly repelled 96.7 % of the rice moths (Corcyra cephalonica) placed in an olfactometer within 30 min at 28 ˚C¹³¹. An ethanol extract produced mild anticariogenic activity against Streptococcus mutans; an MIC value of >10 mg/ mL was reported¹³². Daphneside and daphnorin from them ethanol leaf extract significantly inhibited the receptor activator for nuclear factor-κB ligand-induced osteoclast formation in murine bone-marrow macrophages¹³³. Historical artefacts and traditions of henna use show that women have long applied henna to their feet, hands, nails and hair. The earliest artefact depicting living women with henna stains on the soles of their feet, nails and hands is a wall in Akrotiri, “The House of the Ladies,” Room1, east section, north wall. In this wall painting, both women have henna stains on their soles and fingernails¹³⁴.

32) Toxicology and Safety evaluation:

Reports of the European Commission, formulated by the Scientific Committees on Consumer Products and Consumer Safety^135,136 provide a summary of detailed toxicity studies done on henna, as required for product registration. An acute toxicity study of henna conducted in Sprague-Dawley rats established the median oral lethal dose as above 2000 mg/kg b.w., while an acute dermal toxicity assay in Wistar rats also yielded a median lethal dose of more than 2000 mg/kg b.w.^135,136. In addition, henna did not display signs of irritation potential when tested for acute dermal toxicity. A slight and transient irritation to some individuals were reported in an assay to determine the extent of mucous membrane irritation in New Zealand White rabbits, after exposing the conjunctivital sac of the eyes to henna¹³⁵. No reaction to henna was apparent in skin sensitization experiments in guinea pigs. In addition, there were no indications of any effects of henna in a repeated insult path test on humans. Since henna is applied to the skin and hair, it is important to establish the degree of absorption through the skin. In vitro assays using isolated pig skin mounted in permeation chambers were exposed to henna pulp, containing 1 % lawsone. It was calculated that after exposure of the skin to henna pulp for 30 min and a follow-up period of 72 h, 0.06 % of the applied lawsone remained in the skin¹³⁵. An absolute penetration rate of 703 ng/cm² lawsone was established. Excised, non-viable human skin mounted in flow-through diffusion cells was exposed to henna containing C¹⁴-labelled lawsone in the form of shampoos (5 min exposure) and hair colour pastes (1 h exposure)¹³⁷. In the case of pastes, 2.2 % to 3.7 % of the applied lawsone dose remained in the skin, while 3.6 to 6.8 % of the lawsone from shampoos could be detected To establish whether percutaneous absorption of henna takes place in vivo, aqueous Lawsonia inermis pulp spiked with C¹⁴- lawsone, was placed on the skin of male and female rats for 40 min and then rinsed^135,136. After 72 hours the animals were sacrificed and various body parts were analysed. It was determined that 0.2 % of the applied lawsone was absorbed percutaneously. A bacterial gene mutation assay against five strains of Salmonella typhurium did not reveal any subsequent revertants in the strains tested when exposed to between 50 and 5000 microgram henna per plate^135,136. It was concluded that henna is non-mutagenic under those conditions, since no gene mutations by base-pair changes or frame shifts in genome were induced. In addition, no gene mutations were caused by henna in the mammalian cell gene mutation assay using Chinese hamster cells or using mouse lymphoma cells. However, henna was found to be mutagenic in the tk gene mutation test with mouse lymphoma cells¹³⁶. Incubation of aqueous, methanol, butanol, ethyl acetate, chloroform and hexane leaf extracts with Escherichia coli PQ37 bacteria did not reveal genotoxic effects at concentrations upto 1 mg/assay, since the induction factor remained below 1.5¹³⁸. Henna was found to be weakly clastogenic due to an increase in structural chromosome aberrations in cultured human lymphocytes after exposure to henna^135,136. However, the lack of induced micronuclei in bone marrow of mice treated with 300 mg/kg b.w. henna, a dose known to cause clinical signs and bone marrow toxicity, suggested no indication of clastogenic potential. To determine teratogenic effects, pregnant Sprague Dawley rats were dosed with 40–1000 mg henna/kg/day b.w. from Day 6 to Day 15 of gestation¹³⁵. No clinical signs, no abortions and no mortalities were recorded at all. However, at the highest concentration (1000 mg/kg/day), brain development was impaired in some fetuses and one was born with a cleft palate. At the 200 mg dose, some pubic bone, palate and spinal abnormalities were recorded. The NOAEL (No-Observed-Adverse-Effect-Level) for henna for pregnant rats was set at 200 mg/kg/day and for foetuses at 40 mg/kg/day. Based on the setoxicity assays, of the European Union recommended that henna is safe to use as a hair dye when formulated and applied as indicated¹³⁶. However, commercial henna body art products, often manufactured with adulterants and unlisted compositions are excluded from there commendation due to concerns over mutagenicity. There ports^135,136 are based on a considerable body of evidence indicating very little toxicity posed by pure henna. At present, henna may be imported unconditionally in to the USA when labelled as a hair dye, but the use of henna for body art is for bidden¹³⁹ and customs are empowered to confiscate henna products destined for body art. Most commercial henna body art products are mixtures of henna and other ingredients, often added to darken the stain. These products may pose significant health risks and are illegal for use on skin¹⁴⁰. Henna pastes may contain adulterants such as silver nitrate, carmine, pyrogallol, disperse orange dye, p-phenylenediamine, and even chromium¹⁴⁰. Allergic reactions and chronic inflammatory reactions may result from their use¹⁴¹. Henna hair dye products referred to as ‘black henna’ or ‘neutral henna’ do not contain henna; instead the plant Indigo feratinctoria (indigo) or Cassia obovata, in combination with unlisted p-phenylenediamine dyes and chemicals, may be present¹⁴⁰. Black henna used for tattooing often contains p-phenylenediamine (PPD), an aromatic amine which reacts with components containing at least one aromatic ring in their structures^143-145. A black and long-lasting stain is created in minutes, rather than hours, when PPD mixed with henna is applied in decorative patterns to the skin¹⁴⁶. The use of PPD is the main cause of allergic contact dermatitis or serious allergic reactions from henna tattoo mixtures^{147,148,141,149-152}. Allergic reactions may present as itching, blistering, depigmented skin patches and even permanent scarring, which are treated with antibiotics and corticosteroids^153-157. Apart from the use of PPD, many other chemicals including p-methyl amino phenol, p-amino benzene, p-toluenodiamine, benzene, petrol, butane, hydroquinone and isobutyl p-amino benzoate are added to natural henna and have been associated with contact dermatitis¹⁵⁸. In some cases, the black henna being used for tattooing may contain no henna extract at all¹⁵⁹. Henna extract or lawsone from the plant were found to induce a severe hemolytic anaemia in various animal models after cutaneous exposure or ingestion. This reaction is associated with oxidative damage to erythrocytes^160,161. Allergic reactions may be extremely severe. A 15-year old girl reportedly died of laryngeal edema and pulmonary congestion, consistent with anaphylaxis, following deliberate ingestion of an unknown amount of henna in a suicide¹⁶². Acute haemolysis can take place in G6PD-deficient individuals after applying henna dye to the skin. Observed symptoms are anaemia, reticulocytosis and indirect hyperbilirubinaemia¹⁶³.

CONCLUSION:

The Lawsonia inermis is a multipurpose tree species, widely used for non-food products and medicines. Every part of the L. inermis tree is reported to be useful. The global demand for Lawsonia inermis has increased dramatically as more sectors such as the herbal product Industries. The plant oils have been useful for hair growth applicant, since ancient times. L. inermis leaves, bark, roots and fruits are where found to be show interesting anthelmintic, antioxidant, antidiabetic, anticonvulsant activities and antibacterial activity, trypsin inhibitory activity, abortifacient activities. It is hoped that this review will be a strong stimulus for research or development efforts towards better understanding and utilization of the plant Lawsonia inermis.

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Received on 23.07.2017 Accepted on 29.09.2017

Asian J. Pharm. Tech. 2017; 7 (4): 237-250.

DOI: 10.5958/2231-5713.2017.00036.8

Plant Part	Compounds
	Naphthoquinone derivatives
Leaves	Lawsone (2-hydroxy 1,4-naphthoquinone); 1,3-dihydroxy naphthalene; 1,4-napthaquinone; 1,2- dihydroxy-4- Glucosylnaphthalene
Stem bark	Isoplumbagin
	Phenolic compounds
Bark, Leaves	Lalioside (2,3,4,6-tetrahydroxyacetoxy-2-β-D-glucopyranoside); Lawsoniaside (1,3,4-trihydroxynaphthalene 1,4-di-β-D-gluco-pyronoside); Lawsoniaside B (3-(4-O-a-D-glucopyranosyl-3,5-dimethoxy) phenyl-2E-propenol); syringinoside; daphneside; agrimonolide 6-O-β; D-glucopyranoside; (+)-syringaresinol; O-β-D-glucopyranoside; (+)-Pinoresinol; di-O-β-D-glucopyranoside; Syringaresinol; di-O-β-D- glucopyranoside; isoscutellarin; daphnorin
	Terpenoids
Bark, Seeds	3β, 30-dihydroxylup-20(29)-ene (hennadiol-triterpenes); (20S)-3β, 30-Dihydroxylupane; Lupeol; 30-nor-lupan-3β-ol-20-one; betulin; betulinic acid; lawnermis acid (3β-28β-hydroxy-urs-12, 20-diene-28-oic acid) and its methyl ester
	Sterols
Roots, Leaves, Bark	Lawsaritol ( 24β-ethycholest-4-en-3β-ol); Stigmasterol and β-sitosterol
	Aliphatic constituents
Stem bark	3-methyl-nonacosan-1-ol; n-tricontyl n-tridecanoate
	Xanthones
Whole plant	Laxanthone I (1,3 dihydroxy-6,7 dimethoxyxanthone); Laxanthone II (1-hydroxy-3,6 diacetoxy-7-methoxyxanthone);Laxanthone III ( 1-hydroxy- 6-acetoxy xanthone)
	Coumarine
Whole plant	Lacoumarin (5-allyoxy-7-hydroxycoumarin)
	Flavonoids
Leaves	Apigenin-7-glucoside; apigenin-4-glycoside; luteolin-7-glucoside; luteolin-3-glycoside
Bark	Luteolin-40-O-β-D-glucopyranoside; apigenin; luteolin-7-O-rutinoside; diosmetin-7-O-rutinoside; luteolin-7-O-β-D-glucopyranoside