Matured H. polyrhizus was collected from Patheingyi Township and authenticated. After authentication process, harvested fruits were cleaned and peeled by using Ceramic knife to avoid the interaction between the acidic compound present in the fruit and the steel or metal knife. Then fruit pulp was cut into small pieces and crushed by hands. The betacyanin pigment was extracted by cold maceration with 50% ethanol for 3 days. Then, the extract was obtained by double filtering by muslin. The extract was concentrated by freeze drier and resultant concentrated solution was stored in refrigerator (4˚C) for future use.

Betacyanin content of extract was determined by UV-Visible Spectrophotometer. Betacyanin content was measured in triplicate in de-ionized water. The extract was diluted by 100-fold with deionized water to obtain the absorption value. After 20 minutes of equilibration, the quantification of betacyanin was carried out by applying the following equation [7].

B C = A × D F × M W × 1 0 0 0 E × L

BC = Betacyanin concentration in mg/L

A = Absorption value at the absorption maximum (λ_max= 536nm)

DF = Dilution factor

MW = Molecular weight of betacyanin (550 g/mol)

Ɛ (Molar extinction coefficient of betacyanin (60,000 L∙mol⁻¹∙cm⁻¹ in H₂O)

L = Path length of the cuvette

Each ingredient used in formulation of lipstick was identified by scanning on FT-IR spectrophotometer and the resultant spectra were identified by comparing to the reference spectrum of each ingredient. The spectra obtained were saved for incompatibility study. Before main formulation of lipstick, incompatibility study was carried out by scanning the pre-formulated lipsticks on FT-IR spectrophotometer and incompatibility was checked by comparing the resultant spectrum with initial spectrum of each ingredient.

Melting Point

Formulated lipsticks were completely melted on temperature controlled hotplate. Three capillary tubes were also placed in temperature control oven at 70˚C for 10 mins. The melted lipsticks were poured into the capillary tube while hot and allowed to solidify for 10 mins. Then capillary tubes were placed into melting point measuring device. Melting point determination was done 3 times simultaneously and the melting point ratio was observed.

Surface Abnormalities

Formulated lipsticks were visually checked by an evaluator for surface defects such as crystal formation, contamination by mould and fungi etc.

Aging Stability

The formulated lipsticks were stored in temperature controlled oven at 40˚C for 1 hour. The formulated lipsticks were evaluated for various parameters such as bleeding, crystallization of on surface and ease of application.

pH Parameter

The formulated lipsticks were placed in 25 ml beaker and heated on temperature controlled hotplate at 70˚C. The probe of pH meter was placed into the beaker of melted formulated lipsticks and pH was measured during while hot.

Perfume Stability

The formulated lipsticks were stored at room temperature for 1 month and then evaluated by evaluator for the presence or absence of the fragrance.

The antioxidant activity of extract and formulated lipstick were determined by the DPPH free radical scavenging assay. Briefly, the reaction mixture containing 10 µL of test sample (2, 4, 6, 8, 10 µg/ mL) and 90 µL of DPPH (60 µmol) dissolved in ethanol, was taken in a 96-well micro-titer plate and kept standing at 37˚C for 30 mins. The absorbance was measured at 517 nm by using 96 well microplate-reader and spectrophotometer (Spectrostar Nano, BMG Labtech Microplate Reader). Ascorbic acid was used as standard. Percent radical scavenging activity (%RSA) was calculated by using the following formula;

% R S A = 1 − ( O D T e s t O D C o n t r o l ) × 1 0 0 [8]

%RSA = Percentage Radical Scavenging Activity

OD_Test = Absorbance of test compound

OD_Control = Absorbance of DPPH alone

The IC₅₀ of Standard ascorbic acid, extract and lipstick with betacyanin pigment were calculated from %RSA by GraphPad Prism 5 software [log (inhibitor) Vs. normalized response-variable slope].

Three months old healthy 10 females ddY strain albino mice (mean body weight = 25 - 28 g) were selected. After acclimatization 1 week period, albino mice were divided into 2 groups and mice were performed in test procedure and each group consisted of 5 numbers of mice for experimentation with each test samples. For the test groups of mice, each mouse was lightly anaesthetized with chloroform and approximately 10 mg of lipstick samples were applied to the dorsal portion of left ears of them. Other side of ears serves as an untreated control. Daily application is carried out up to 4 successive days and checked assessment of irritation. Assessment was carried out immediately before applications were made during the first 4 days of experiment. On the fifth day, assessment was carried out 24 hour after the last (fourth) application of test samples. The daily difference between negative control ear and treated ears for each animal are summed to give an overall total for all the mice in an experiment. A correction is made for any difference between control and treated ears initially. The total, divided by 5, yields a mean for each test material for the experimental period [9].

Microbial analysis was carried out to ensure the microbial safety of formulated lipsticks for customers to use and to confirm hygienic and high quality handling. The microbial analysis of the samples was determined by spread plate method by modifying method of Hitchins and colleagues. Briefly, 1 g of the sample was diluted with the sterilized normal saline solution to get 1:10 dilution and the diluted samples were mixed thoroughly. 0.5 mL of each of the diluted samples were pipetted onto the surface of a solidified agar medium and spread evenly with a sterilized glass spreader rod. After spreading, the plates were incubated at 37˚C for 48 hrs. For total combined yeast and mold count, the plates were incubated at 25˚C and observed daily for 7 days. After incubation period, the colonies grown on the plate were counted and the number of bacteria in the original sample was calculated by multiplying with the dilution factor. The results were reported as cfu/g (mL) sample [10]. The following different Media were used for the determination of presence or absence of different pathogenic microbes in the samples.

1) Nutrient Agar for total aerobic microbial count

2) Potato Dextrose Agar (PDA) for total combined yeast and mold count

3) Mannitol Salt Agar (MS) for Staphylococcus aureus

4) Cetrimide Agar for Pseudomonas aeruginosa

5) Eosin Methylene Blue Agar (EMB) for Escherichia col.

One gram of lipstick was accurately weighed on a digital balance. The lipstick was digested by triacidic mixture (70:7:23) of Nitric acid, Sulphuric acid and Perchloric acid on electronically temperature controlled hotplate until white fume was obtained. The digestion was carried out at low temperature at first followed by increase in temperature. The digest was cool and quantitative amount of de-ionized water was added and was filtered into 10 mL volumetric flask. After cooling, the final volume was adjusted volumetrically with de-ionized water [1]. Sample solutions were freshly prepared by digestion method. The resultant solutions were injected into Flame AAS and lead contents were determined at optimized condition by proposed method. All the measurements were triplicate. The results of sample were expressed as part per million (ppm).

Data obtained from the general evaluation of formulated lipsticks revealed that the formulated lipsticks had the acceptable quality (Table 2). The formulated lipsticks had appropriate organoleptic characteristics (color, odor and appearance). It was observed that there was no sweating, bleeding, streaking and blooming after one week of observation, when the products were store in room temperature for one month and oven temperature (40˚C) for an hour. The melting point of formulated lipstick was less than 60˚C. The pH value of formulated lipstick was very near to neutral pH 7 and no surface abnormalities, no visible sign of mould and fungi. Aging stability test also showed that formulated lipstick had suitable physical quality requirement.

The 50% inhibitory concentration for free radical (IC₅₀) of Standard ascorbic acid was 4.51 µg/ml. The IC₅₀ value of ethanolic extract of Red dragon fruit, lipstick with betacyanin pigment were 10.28 and 22.23 µg/ml respectively. % RSA on DPPH radical at five serial concentrations of Standard ascorbic acid, Red dragon fruit extract, lipstick with betacyanin pigment were described in Figure 4.

Azwanida and his colleagues reported that 50% inhibitory concentration (IC₅₀) of formulated lipstick with betalanin pigments extracted from RDF was 54.29 ± 1.9 μg/ml and IC₅₀ value of the formulated lipstick (without pigment added) was 76.31 ± 0.43 μg/ml while the IC₅₀ value for the reference ascorbic acid was 14.56 ± 0.24 μg/ml [3].

The present study showed that IC₅₀ values of red dragon fruit extract was approximately 2 times greater than those of standard ascorbic acid. Accordingly, the antioxidant activity of Red dragon fruit was satisfactorily high. High antioxidant activity of extract was mainly due to the presence of betacyanin compound. The IC₅₀ value of lipstick was greater 2.3 times greater than those of RDF may be due to the dilution of extract with other ingredients and exposure to relative high temperature 70˚C during formulation process.

The responses obtained from the Mouse ear irritancy test also less than 9 (Table 3). The statistical analyses of variance were performed by ANOVA with Microsoft Office Excel 2010 for Windows and P value less than 0.05 considered as statistically significant. The formulated lipsticks were not statistically significant from control ears in mouse ear irritancy test. Therefore it can be concluded that the formulated lipsticks have negligible skin irritancy.

In many studies on formulation of herbal lipstick, skin irritation test was carried out on 10 human volunteers. With new cosmetic ingredients, laboratory animal studies should invariably be carried out before they are tested on human skin; even with well-known raw materials, it is prudent to screen new formulations similarly. Therefore, mouse ear irritancy test was carried out in present study instead of human volunteers.

Formulated lipstick was products of category 2 and microbial analysis of lipstick sample proved that these lipsticks was microbiological suitable for cosmetics use. In quantitative analysis for microbiological control, lipsticks with betacyanin

Interpretation for Final response; <9 = not perceptibly irritant, 10 - 15 = slightly irritant, >15 = sufficiently irritant.

pigment had no visible colony. Yeast and mould were not detected in 0.5 g of both formulated lipstick sample. Moreover in qualitative analysis, Staphylococcus aureus, E. coli and Pseudomonas aeruginosa were not detected in 0.5 g of formulated lipstick.

Microbial analysis of lipstick with color extract of Red dragon fruit was satisfactory. No bacterial, mould and yeast, fungi were detected. Potentially pathogenic microorganisms such as Staphylococcus aureus, Pseudomonas aeruginosa and E. coli were not detected. This may be due to antibacterial activity of ethanolic extract of Red dragon fruit and preservative do not require for lipstick with color extract of Red dragon fruit within the study period. This pointed out the antimicrobial activity of RDF extract. This finding was consistent with the research finding of Azwandia in Malaysia that the pigments of Red dragon fruit possess potent antimicrobial activity.

The present study found formulated lipstick that using betacyanin pigment as color ingredient had very low level of lead. The mean lead content in lipstick with betacyanin pigment was only 2.88 ± 0.09 ppm. In contrast to the market sample lipsticks, mean leads content of natural color lipsticks was very lower than mean lead content of Counterfeit, Fair Price, Regular Price, Less Expensive and Expensive lipstick groups (Figure 5). Very low level of lead was detected. Detected amount of lead in these formulated lipsticks with natural color are lower than the specified limit of FDA (20 ppm) and limit of Health Canada (10 ppm) [12] [13]. Form the result of this study showed that natural color compound can reduce the lead content of lipstick.