3. Chemical Constituents and Phytochemical Screening of Thanaka
|Scientific Name and Parts||Solvent|
|Instrumental Analysis and Characterizations Tests||Results||References|
|Stem bark||Methanol and chloroform (1:1)|
Evaporated extract purified by silica gel chromatography, eluted with chloroform.
|Thin-layer chromatography (TLC) performed with 40:1 mixture of chloroform and methanol.|
Further purification done through silica gel chromatography and 2nd TLC performed to obtain fraction with strongest fluorescence spots.
Fractions then purified using reverse-phase High-performance liquid chromatography (HPLC) and dried to obtain crystallized active compound by Mass Spectrometry (MS) analysis.
|Active compound characterized as marmesin (2,3-dihydro-2-(1-hydrozy-1-methylethyl)-furanocoumarin), which tested to be able to absorb wide range of UV-A radiation (λmax at 335 nm).|||
|Petroleum||Extract was purified by chromatography on neutral alumina, then rechromatography and recrystallization.|
Crystalline compound was analysed through nuclear magnetic resonance (NMR) spectroscopy.
|Alkaloid compound identified as 4-methoxy-1-methyl-2-quinolone.|||
|Petroleum||Solvent chromatographed on neutral alumina.|
Crystalline compounds were analysed through NMR spectroscopy.
|Extracted with 70% acetone to obtain the crude and suspended in water, then repeated extraction using ethyl acetate||Compounds identified:|
N-Benzoyltyramine methyl ether
3 β-Hydroxy-5α,8 α-epidioxyergosta-6,22-diene
|Ethanol||Gas chromatography Mass spectrometry (GC-MS) analysis||20 phytocomponents identified in bark ethanolic extract, namely:|||
|Stem bark & Leaf||Crude powder, no extraction was performed.||Phytochemicals|
|Phytochemicals found in Leaf:|
|Phytochemicals found in Stem bark:|
|Present in leaf:|
|Present in stem:|
Present in bark:
|High-performance Thin Layer Chromatography (HPTLC) profiling||Leaf|
8 peaks with Rf values in the range of 0.09 to 0.49
10 peaks with Rf values in the range of 0.08 to 0.65
8 peaks with Rf values in the range of 0.07 to 0.63
|Leaf||Ethanol||GC-MS analysis||16 phytocomponents in leaf ethanolic extract, namely|||
|Hot extraction method using Soxhlet apparatus by mixing powdered leaf in ethanol.||GC-MS analysis||27 compounds identified in leaf extract, namely|||
|Acetone||GC-MS analysis||8 compounds were found, namely|||
Acid and alkaline treatment
Ferric chloride/sulfuric acid
Saponin & Flavonoid:
Ascorbic acid content:
|Fibres (47 g/kg)|
Phytosterols (38.7 g/kg)
Polyphenols (67.4 g/kg)
Flavonoids (0.6 g/kg)
Saponins (0.18 g/kg)
Ascorbic acid (0.54 g/kg)
4. Biological Properties of Thanaka
4.1. Antioxidant Activity
4.2. Antimicrobial Activity
4.3. Cytotoxicity and Cell Viability
4.4. Other Biological Properties
5. Cosmeceutical Products Containing Thanaka in the Southeast Asia Market
6. Conclusions and Perspectives
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Conflicts of Interest
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|“Limonia Acidissima L.” OR “Hesperethusa crenulata” OR “Naringi crenulata” OR “Thanaka” OR “Limonia crenulata”|
|Scientific Name and Parts||Solvent Extraction||Biological Assays||Results||References|
85% Aqueous ethanol
|Diphenyl-picrylhydrazyl (DPPH) radicals scavenging||85% ethanol > methanol = ethyl acetate > water > dichloromethane ≥ hexane (highest to lowest)|
|Total Phenolic Content (TPC)|
|Folin–Ciocalteu reagent||Methanol > ethyl acetate > 85% ethanol > dichloromethane > water ≥ hexane (highest to lowest)|
|Tyrosinase Inhibition Activity|
|Kojic acid as positive control||Dichloromethane > hexane > ethyl acetate > 85% ethanol > water > methanol (highest to lowest)|
Original bark powder also showed very mild tyrosinase inhibition activity, may be due to colour of powder affected microplate reading.
Tyrosinase inhibition activity of the Thanaka extracts are mild as compared to kojic acid.
|Minimum inhibitory concentration (MIC) assay and|
Minimum bactericidal concentration (MBC) assay
Staphylococcus aureus and Escherichia coli
|All extracts and original bark powder possessed a slight antibacterial activity that is 10-to 20-fold lower activity against Escherichia coli (E. coli) and a 300-fold lower activity against Staphylococcus aureus (S. aureus), when compared with clindamycin.|
|Cytotoxicity using MTT Assay|
|Cell line used:|
human melanoma A-375 cell line
|Dichloromethane (0.30 ± 0.01) < hexane (0.48 ± 0.01) < ethyl acetate (0.90 ± 0.01) < 85% ethanol (12.81 ± 0.16) < methanol (15.30 ± 0.20) < water (19.07 ± 0.49) (highest to lowest). IC50 of standard doxorubicin = 0.0003 ± 0.00|
Original bark powder at 15 mg/mL showed no detectable cytotoxicity effect on A-375 cells.
|Cell line used:|
Murine macrophage like cell line RAW 264.7
|Hexane > dichloromethane > ethyl acetate > 85% ethanol > methanol > water (highest to lowest)|
All extracts possessed 80–90% high anti-inflammatory activity at non-toxic concentrations (80% cell viability)
|Genotoxicity using Comet assay|
|Cell line used:|
A-375 cell line
Concentrations of samples:
Those that showed 95% cell viability from MTT assay
|All six extracts and original bark powder exhibited no genotoxicity, while obvious DNA strand breaks were observed in cells treated with very low concentrations of hydrogen peroxide.|
Leaf powder was added into double distilled water, boiled in water bath follow by filtration to acquire pure extract.
Tin nanoparticles (SnO2 NPs) synthesis:
Tin chloride was mixed in the leaf extract, heated in a water bath to obtain paste, followed by calcination to obtain dark grey coloured dry material, and grinded to powder for further use.
|Cell Viability Test using MTT assay|||
|Cell line used:|
Human cervical cancer (SiHa) cell line
Positive control used:
Cisplatin (10 μg/mL)
Concentrations of samples:
SnO2 NPs (20–200 μg/mL)
|Cell viability of SiHa cells reduced in a concentration-dependent manner of SnO2 NPs. Concentration of SnO2 NPs at 35 ± 0.03 μg/mL was fixed as effective dose.|
|Morphological Assessment of Apoptosis|
100, 200, 300, 400, 500, 600, 700, 800 and 900 mg/L
|Acetone (1.020 mg/L) > methanol (1.134 mg/L) > ethyl acetate (1.815 mg/mL) > hexane (9.744 mg/L) (highest to lowest)|
|Seed||Preparation of defatted seed flour:|
Seeds were grounded to fine powder, removed fat by soaking and stirring in petroleum ether, and then decanted solvent (Repeat 3 times). Seed flour then air dried.
Protein hydrolysates preparation:Defatted seed flour was suspended in alkali extraction, then centrifuged to obtain supernatant. Supernatant then further precipitated at pH 4, washed thrice and dried in vacuum oven.
|DPPH assay was found not a suitable assay for protein hydrolysates. As the concentration increases, precipitation occurred; subsequently, the cloudiness affects the antioxidant activity measurement.|
In ABTS assay, the scavenging activity % of protein hydrolysates are observed to be increasing as the concentration of peptides increases and results in higher scavenging activity % compared to Trolox.
In FRAP assay, the absorbance of FRAP did increase when the concentration increases, but results much lower compared to Trolox.
In metal chelating ability assay, the activity % increases as the concentration increases and the % is observed to be higher than Trolox.
|Protein hydrolysates preparation:Defatted seed flour was suspended in alkali extraction, then centrifuged to obtain supernatant. Supernatant then further precipitated at pH 4, washed thrice and dried in vacuum oven.||Storage Stability|||
|Concentration of L. acidissima protein hydrolysates (LAPH): ranging from 0.04 to 2.00%|
Added to 5% crude anthocyanin extract and stored in the presence of light for 0–5 days.
|Preservation of anthocyanin was observed to slightly increase from 2.81 days to 3.25 days at 0.12% LAPH concentration but decrease to 3 days at concentration 2.00%.|
|Colour Stability and Fading of Anthocyanin|
|pH differential method:|
Red intensity measured at absorbance 523 nm.
|Colour stability of anthocyanin was observed to be higher when treated with 2.00% LAPH, while the colour stability also increase as the concentration of LAPH increases at different concentrations, as compared to non-treated anthocyanin.|
|Regulation of Fluoride-Induced Hyperglycaemia and Hyperlipidaemia|||
Colony-bred male albino rats
NC (Normal control)
FC (fluoride control)
F LA I (fluoride-exposed given 2.5 g/kg fruit powder in feed)
F LA II (Given 5 g/kg in feed)
F LA III (Given 10 g/kg in feed)
|FC groups showed a significantly increase plasma glucose levels, G-6-Pase activity and a significantly decreased hepatic glycogen content and hexokinase activity while fruits powder treated groups F LA (I, II, III) led to a significant decrease in plasma glucose levels and G-6-Pase activity, while an increase in hepatic glycogen content and hexokinase activity in a dose-dependent manner.|
LA treatment also increased in a dose-dependent manner in the plasma HDL-C content and decreased plasma TL, TC, TG, LDL-C, VLDL-C and AI content in fluoride-exposed mice.
Dose-dependent decrease of hepatic lipid profiles was observed in LA-treated fluoride-exposed mice.
Extracted crude extract fractioned into:
|Brine shrimp used:|
Positive control used:
|Vincristine sulphate (0.451 μg/mL) > petroleum ether (2.0779 μg/mL) > chloroform (6.8975 μg/mL) > methanolic (20.6226 μg/mL) (highest to lowest)|
|DPPH radicals scavenging|
Butylated hydroxy toluene (BHT)
|BHT (17.69 μg/mL) > chloroform (18.8 μg/mL) > petroleum ether (37.64 μg/mL) > methanolic (292.16 μg/mL) (highest to lowest)|
Four herbs powder including Thanaka bark powder were mixed, wrapped in muslin cloth, dipped in water and steamed over hot water bath. Crude extract was squeezed from the herbal ball, then centrifuged, filtered supernatant and finally, freeze dried.
|DPPH radicals scavenging|
Gallic acid as control
|Overall antioxidant activity of herbal ball extract (219.27 ± 36.98 μg/mL) is lower than gallic acid (4.97 ± 0.16 μg/mL) by 40-fold.|
|Antibacterial Activity using MIC and MBC assay|
|Cell line used:|
Propionibacterium acnes (P. acnes)
Tetracycline (0.24 to 0.0075 μg/mL) as control
|The herbal ball extract showed anti P. acnes activity with the MIC value equal to the MBC, which is 31.25 μg/mL.|
The activity of herbal ball extract was lower by 500-fold as compared to tetracycline (MIC= 0.06 μg/mL).
Chopped leaf was mixed in 50 mL of deionized water, boiled over microwave oven for 10 min, then cooled and filtered.
Synthesis of silver nanoparticles (AgNPs):
Aqueous extract was added into aqueous solution of silver nitrate, AgNO3 (1 mM), shaked to mix and kept in dark conditions until noticeable colour change.
Formation of AgNPs was monitored by UV-Vis spectral scanning in 300–700 nm range.
|Wound Healing Activity|||
|Formulation of ointment:|
Batch A: simple ointment without any treatment (control)
Batch B: Betadine (5% w/w) (standard drug)
Batch C: Silver nanoparticles (5% w/w) synthesized using Thanaka leaf extract.
In vivo wound healing activity
Animal model: Wistar male albino rats
Wound model preparation:
One-excicion wound inflicted by cutting 500 mm2 full thickness of skin from back of rats
Treatment Group I:
Negative control. Treated with Batch A ointment.
Treatment Group II:
Treated with standard ointment Batch B.
Treatment Group III:
Treatment with AgNPs ointment Batch C.
|Thanaka leaf mediated extract AgNPs > betadine (standard) > no treatment (Negative control) (highest to lowest)|
|Country||Brand||Type of Products||Ingredients||Functions||References|
|Myanmar||Shwe Pyi Nann||Export Shinmataung Thanaka powder||Made with Thanaka bark and Myanmar medicinal herbs||Protects skin from UV damage, provides anti-aging effect.|||
|Kant Kaw Stamen Thanaka powder||Made with kan kaw stamen and Thanaka bark||Provides UV protection anti-bacterial, anti-fungal, anti-pigmentation and anti-aging effect, with skin cooling sensation and fragrance.|
|Thanaka with lime powder||Thanaka bark with lime||Provides acne-clearing, anti-pigmentation and skin soothing effect.|
|Whitening Thanaka powder||Thanaka bark with lime||Provides acne-clearing and anti-pigmentation effect. With fragrance.|
|Jasmine Thanaka powder||Thanaka powder with jasmine fragrance|
|Rose Thanaka powder||Thanaka powder with rose fragrance|
|Star flower Thanaka powder||Thanaka powder with star flower fragrance.|
|Thanaka Aloe vera body lotion||Thanaka powder with aloe vera||For anti-aging, acne treatment, moisturizing and UV protection with skin soothing effect from aloe vera.|
|Thanaka Jasmine body lotion||Thanaka powder with jasmine||For anti-aging, acne treatment, moisturizing and UV protection with jasmine fragrance.|
|Thanaka Kant Kaw Stamen body lotion||Thanaka powder with kant kaw stamen.||For anti-aging, acne treatment, moisturizing and UV protection with whitening effect and fragrance from kant kaw stamen.|
|Thanaka Lime body lotion||Thanaka powder with lime||For anti-aging, acne treatment, moisturizing and UV protection with whitening effect and fragrance from lime.|
|Thanaka original body lotion||Thanaka powder||For anti-aging, acne treatment, moisturizing and UV protection.|
|Thanaka Rose body lotion||Thanaka powder with rose.||For anti-aging, acne treatment, moisturizing and UV protection with whitening effect and fragrance from rose.|
|Thanaka Shimmataung Root body lotion||Thanaka powder with Shimmataung root.||For anti-aging, acne treatment, moisturizing and UV protection with whitening effect from Shimmataung root.|
|Thanaka Star flower body lotion||Thanaka powder with star flower||For anti-aging, acne treatment, moisturizing and UV protection with fragrance from star flower.|
|Thanaka pudding face cream||Thanaka powder with rose, jasmine, star flower, aloe vera, kant kaw stamen and lime||Moisturizes skin, acne-clearing, whitening, anti-pigmentation and anti-aging effect.|
|Snake brand||Wild Thanaka cooling powder||Thanaka powder||Provide cooling sensation on skin, controls sweat and oil on skin to prevent rashes and acne.|
|Pann Chit Thu||Thanaka perfume block||Thanaka powder||Rub with clean water and apply as face mask. Provides anti-aging, smoothens the skin, whitening and UV-protection.|
|Myat Bhoon Pwint||Whitening Thanaka lotion||Thanaka powder||Provides skin with moisture and whitening of skin.|
|Truly Thanaka||100% Pure thanaka powder||Thanaka powder||For acne treatment, anti-pigmentation, enhance skin complexity, anti-aging.|||
|Royal youth Thanaka anti-aging treatment lotion serum||Thanaka powder||Anti-aging and antioxidant. Provides skin with moisture and cooling effect for post-sun exposure.|
|Pure Thanaka & 24 K gold face mask||Thanaka powder with 24 K gold||Added 24 K gold to enhance anti-aging properties.|
|Bamboo charcoal & Thanaka deep cleanse face mask powder||Thanaka powder with bamboo charcoal||Provides deep cleansing to heal acne, brightens skin and shrinking pores for firming of skin.|
|Colloidal oatmeal & Thanaka face mask powder||Thanaka powder with colloidal oatmeal||Helps relieve skin irritations caused by eczema and skin rashes.|
|Jasmine rice & Thanaka detoxifying mask||Thanaka powder with jasmine rice||Helps to detoxify, soothes skin for post-sun exposure. In addition, brightens skin and reduce dark circle appearance.|
|Kaffir lime, honey & Thanaka soap||Kaffir lime, honey and Thanaka||For gentle cleansing, anti-acne and moisturizing of skin.|
|Acne + blemish clearing night serum||Thanaka extract with vitamins A, C and E||For anti-aging, anti-acne, removes skin blemishes and even skin tone.|
|Thailand||Supaporn||Thanaka powder||Thanaka powder||General powder for multiple uses as face masks or body scrub, provides oil control, moisturizing, acne clearing and whitening effect.|||
|Thanaka soap||Thanaka powder||Cleanses and exfoliate skin for whitening and softening of skin.|
|100% pure Thanaka powder||Thanaka powder||Uses as facial and body scrub or face and body mask.|
Whitening of skin, anti-oxidant, anti-aging, oil control, acne treatment, moisturizing and UV-protection.
|Herbal scrub||Thanaka powder and collagen||Formulated with Thanaka and collagen to exfoliate for brighter skin and moisturize skin.|
|Herbal face scrub||Thanaka powder||Exfoliates for brighter skin.|
|De Leaf||Loose powder||Thanaka powder||Formulated to control sebum, conceal dullness, supress occurrence of sweat and rashes, while also provides a cool sensation and sense of fragrance when used. The loose powder can also be used as a face mask for smooth and clear skin when mix with water.|||
|Foundation powder||Thanaka powder with vitamins C & E||Provides SPF 20 PA +++ for UV protection, oil control, formulated with vitamins C and E for anti-pigmentation, anti-aging and moisturize skin.|
|Duo translucent loose powder||Thanaka powder and pomegranate powder||Helps to control facial oiliness prior to face makeup, prevents acne and anti-aging.|
|Moisturizing & whitening cream||Thanaka extract with vitamins A, C, E and aloe vera extract||Formulated with vitamins A, C, E and aloe vera extract to nourish, moisturize skin, anti-aging, anti-pigmentation and repairs skin damage from UV rays.|
|White & Smooth body serum||Thanaka extract||Nourishes and hydrates skin, with whitening effect.|
|Face mask||Thanaka extract||For anti-aging and brightening of skin.|
|Soap||Thanaka powder||Gentle exfoliator with moisturizing, anti-aging and skin brightening effect.|
|Malaysia||ThanakaMalaysia||Powder as face mask||Thanaka powder||Multi-purpose powder acting as face masks to prevent acne, dark spot treatment and traditional application as sunscreen.|||
|Permanent hair removal||Thanaka powder||Exfoliation.|
It is sold in package containing a bottle of 100 mL safflower oil and 25 g Thanaka powder.
|Handmade Soap||Thanaka powder||Available in three types (original, treatment and hydrating soap) to cater for different skin types including acne prone skin, dry skin and normal skin types.|
|GloSkin series-Face serum||Hyaluronic acid, Thanaka extract and rose water scent||Formulated with hyaluronic acid, Thanaka extract and Ross water scent.|
For anti-aging, acne treatment, remove skin blemishes, unblock pores and evens skin tone.
|GloSkin series-Face mask||Thanaka powder, glutathione powder and turmeric||Formulated with Thanaka powder, glutathione powder and turmeric.|
For detoxifying, acne removal, even out skin tone and reduce appearance of dark eye circles.
|Bio Essence||Face cleanser||Thanaka extract||Deep cleansing and whitening.|||
|Skin refiner||Thanaka extract||For double cleansing, promote absorption of subsequent skin care products, pore tightening and skin whitening.|
|Skin serum||Thanaka extract||Skin whitening and anti-pigmentation.|
|Day cream||Thanaka extract||Provides moisture, anti-pigmentation and SPF 20 for UV-protection.|
|Night cream||Thanaka extract||Provides skin moisturization, anti-pigmentation and repairs skin.|
|Spot corrector||Thanaka extract||Lighten dark spots and anti-pigmentation.|
|Face mask||Thanaka extract||Moisturising and whitening.|
|Tone up cream||Thanaka extract||Skin whitening and conceals blemishes for instant tone-up effect.|
|Taté Skincare Malaysia||Pure Thanaka Collagen Gold face cream||Thanaka extract with collagen||Provides anti-aging effects.|||
|Organic pure Thanaka face cream||Thanaka extract||Provides anti-acne, anti-aging effect and smoothens skin.|
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