JURNAL DYE Pewarna - Basella Alba Fruit - 2

104Chatchawal et al. IJPS Vol. 6, No.3, Sep-Dec 2010

Original Article

Physical and Biological Properties of Mucilage from Basella alba L. Stem andIts Gel Formulation

, , , , Chuennapa Chatchawal, Natsajee Nualkaew*, Srisomporn Preeprame, Supatra Porasuphatana, Aroonsri Priprame

Received: 8 Apr 2009 Accepted: 20 Sep 2010

(Basella alba Linn) Basellaceae

Swelling capacity 4.33 + 0.29 mL/g dry weight pH 5.3-5.4 Anionexchange chromatography Thin layer chromatography DPPH scavenging assay(IC50 = 514.41 g/mL) (Chang liver cell) 2 mg/mL 84.4%

Chuennapa Chatchawal, 5th year student, Faculty of Pharmaceutical Sciences, Khon-Kaen University, Khon-Kaen 40002,Thailand.

( , 5 . . 40002)*Natsajee Nualkaew, Ph.D., Lecturer, Faculty of Pharmaceutical Sciences, Khon-Kaen University, Khon-Kaen 40002,

Thailand.( , .., , . . 40002)Srisomporn Preeprame, Ph.D., Assistant Professor, Faculty of Pharmaceutical Sciences, Khon-Kaen University, Khon-Kaen

40002, Thailand.( , Ph.D., , . . 40002)Supatra Porasuphatana, Ph.D., Assistant Professor, Faculty of Pharmaceutical Sciences, Khon-Kaen University, Khon-Kaen

40002, Thailand.( , Ph.D., , . . 40002)Aroonsri Priprame, Ph.D., Associate Professor, Faculty of Pharmaceutical Sciences, Khon-Kaen University, Khon-Kaen 40002,

Thailand.( , Ph.D., , . . 40002)*corresponding author, E-mail : [email protected], telephone : 66 43 362092, Fax : 66 43 362092

Physical and Biological Properties of Mucilage from Basella alba L. Stem and Its Gel Formulation105

: , , , , 2553; 6(3): 104-112

AbstractBasella alba Linn is a succulent mucilage plant in Basellaceae family. Its mucilage is the water soluble

polysaccharide. The aqueous extracts from stem of Basella alba Linn were investigated for the general physicalproperties and pharmacological activities. Lyophilized mucilage extract was hygroscopic and could swell in waterwith a swelling capacity of 4.33 + 0.29 mL/g dry weight. Its aqueous solution was viscous with pH 5.3-5.4 andfilm forming upon drying. The TLC analysis of hydrolyzed polysaccharide which was purified by using anionexchange chromatography yielded D-galactose as the major component. Basella alba extract presentedantioxidant activity by DPPH scavenging assay (IC50 = 514.41 g/mL) but not antityrosinase. The mucilageextract 2 mg/mL exhibited relatively mild toxicity to Chang liver cell with 84.4% cell viability. Preliminary gelformulations from Basella mucilage for further development as cosmetic and topical medicine showed goodphysical properties and stability.

Keywords : Basella alba Linn, Mucilage, Polysaccharide, Antioxidant activity, Cell viabilityIJPS 2010; 6(3): 104-112

IntroductionBasella alba Linn. (Indian Spinach, Phak Plang

Khaw, Phak Plang Daeng) belongs to familyBasellaceae with mucilage contained in most parts ofthe whole plant. In Thailand, there are 2 distinctivetypes of B. alba as the colors of the stem appeared inred and green. However, they are classified to be thesame species of B. alba which B. rubra is its synonym(Smitinand, 1992). This plant serves as a Thaitraditional vegetable. The fruit provides dark violet colorfor food colorant. Basella mucilage has been used inThai traditional medicine as topical application forirritant, bruise, ringworm, and laboring. Stem and leavesare used as mild laxative, diuretic, and antipyretic(Chote-Anan et al, 2550). In India, it has been usedfor antipruritis, and burn (Saikia et al, 2006), andhas been used in Bangkladesh for acne and freckletreatment (Akhter, 2008). The Ayurvedic treatmentin India has been used B. alba leaves and stem foranticancer such as melanoma, leukaemia, and oralcancer (Premalatha and Rajgopal, 2005). B. alba

contains basellasaponins (Toshiyuki, 2001), aminoacid such as arginine, leucine, isoleucine, lysine,threonine and tryptophan (Khare, 2007), peptide,phenolic compounds in 95% extract (15.5 mg GAE/g DW) (Maisuthisakul et al 2008). Basella fruitcontains gomphrenin derivative which is betalainpigment (Glassgen et al., 1993). The mucilage ofB. alba consists of mixture of polysaccharides2.6-5.35% (Palanuvej et al 2009; Lin et al 2009);and starch-type glucan which can be separated bystarch iodine complex (Haq et al., 1969). Plantmucilage composes of water soluble polysaccharide.It functions as water retention, germination, foodreservoir and secondary metabolite storage. The stickyproperties of mucilage can be used as medicine andcosmetic proposes, for example marshmallowmucilage is used for cough relief; Psyllium mucilage isused for bulk laxative; the mucilage from okrapossesses stomach cytoprotective activity and gastricreduction (Jadhav, 2008); Aloe vera mucilage is usedas antiinflammation, UV protective, wound healing, and


moisturizer (Dracelos, 2001); mucilage from yam(Dioscorea batatas) is used as skin lubricant, and italso contains allantoin and allantoic acid thatpromoted wound healing and anti skin cancer (Fu etal, 2006). For the pharmaceutical aid, the mucilagecan be used as thickener, water-retention agent,gelling agent, suspending agent, and film former (Janiet al, 2007). To various properties and usages ofplant mucilage, Basella mucilage was also proposedfor those applications of medicine and cosmetics. Thisstudy was to detect for some physical properties aswell as the sugar composition of the Basellamucilage. The antityrosinase which was accorded totraditional used for anti freckle was tested. Antioxidantwas related to the inflammation mechanisms whichcaused by free radical was assay. Then formulation ofgel from Basella mucilage was prepared. This studycan be used for further developing as topical use forcosmetic and anti-inflammatory purposes.

Material and methods1. Plant materialBasella alba L. was purchased from local

market, Khon Kaen province, Thailand. The plant wasidentified by Assistant Prof. Dr. Srisomporn Preprame.The voucher specimen was kept at the Faculty ofPharmaceutical Sciences, Khon Kaen University, KhonKaen, Thailand.

2. Chemical and instrumentsD-glucose anhydrous (Univar, Australia),

D(+)raffinose pentahydrate (Himedia, India),D(+)mannose (Himedia, India), D(+)xylose (Himedia,India), L(+)rhamnose (Himedia, India), D(-)fructose(Fisher Chemicals, UK), D(+)galactose (Univar,Australia), L(+)arabinose (Calbiochem, Germany),D-sorbital (Fluka, Germany), DPPH (2,2-diphenyl-1-picrylhydrazyl) (Fluka, Germany), L-DOPA(Sigma, USA), mushroom tyrosinase (Sigma, USA),MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromine (Molecular Probes, USA), DEAE

650 M (Toyopearl Japan), TLC SiO2 GF254precoated plate (Merck, Germany), freeze dryer(Flexi-Dry, UK), UV-VISIBLE spectrophotometer(Shimadzu UV-1700, Japan), dialysis bag MWCO12000-14000 (Cellu Sep, France), micro platereader (Bio-Rad, USA), rheometer model DV-111programmable (Brookfield, UK).

3. Extraction of mucilage500 g Fresh stems were cleaned, extracted

twice by homogenization in water at the ratio of 1:1,boiled, and filtered through the cheesecloth. Thecombined aqueous extract was lyophilized to drynessand kept in the desiccator until used.

4. Determination of the physical properties ofBasella mucilage

The physical properties of mucilage weredetermined, such as appearance, color, flavor, andpH. The swelling test was performed by bed volumetechnique (Kuniak and Marchessault, 1972). Waterwas gently added into 0.2 g dried Basella mucilageuntil reached the final volume of 5 mL and keptovernight at room temperature. The swelling capacitycould be obtained from the swelling volume (ml)divided by Basella mucilage (g). Film forming wasobserved from spreading 6.7% aqueous mucilage(1 g dried Basella mucilage in 15 ml water) onto thepetri dish 10 cm diameter and incubated at 50 oC forone day. The viscosity was measured by usingrheometer, spindle number 31, RPM 60.0, 30 secper cycle at room temperature.

5. Partial purification of polysaccharide fromBasella mucilage Dried Basella mucilage extract 450 mg wasredissolved in 30 mL water. The crude polysaccharide was precipitated by adding 70 mL ethanol toobtained 70% v/v ethanol final concentration, andplaced overnight at room temperature. The pellet wascollected by centrifugation at 4,000 rpm for 10 minat room temperature, and washed with ethanol. Thenit was redissolved with 120 mL water, and applied


onto DEAE 650M which was pre-equilibrated withwater. The column was eluted by 50 mL of 0, 0.5,1.0, 1.5, and 2 M NaCl in water, respectively. Thefractions of 15 ml were collected. Each fraction wasdetected for protein by UV at 280 nm, and assayedfor the total sugar content by phenol-sulfuric acidmethod. The sugar containing fractions were pooled,dialyzed against water, and lyophilized to dryness.

6. Phenol-sulfuric acid method0.2-mL Sample was mixed with 0.8 mL conc.

H2SO4, then 0.6 mL of 5% aqueous phenol wasadded. The mixture was detected by UV at 490 nm.

7. Determination of monosaccharidecomposition in polysaccharides

One mg of the partial purified polysaccharidefrom B. alba was hydrolyzed with 1 mL 10% HCl,and boiled for 2 hr. The reaction mixture was thenapplied onto SiO2 TLC using 3 mobile phase systemsof CH3CN:H2O (8.5:1.5), CH3CN:EtOH (8:2), andCH3CN:EtOH (13:2), and detected by spraying with10 % aqueous H2SO4, and heated. The Rf value ofauthentic monosaccharides was compared with thatof acid-hydrolyzed sample.

8. DPPH radical-scavenging assayDPPH radical-scavenging assay was

performed in 96-well plate according to Chan et al,2008. Ascorbic acid was used as positive control.The Basella mucilage was diluted with water intoconcentrations of 0.125, 0.25, 0.5, 1, 2.5 mg/mlbefore reacting with 0.25 mM DPPH in MeOH in aratio 1:1 for 30 min. Mixture was protected from lightat room temperature, and measured for UVabsorbance at 550 nm. The percentage of radicalscavenging was calculated:

When ADPPH is absorbance of DPPH in MeOH;Asample is absorbance of mixture of Basella mucilage,and DPPH; Ablank is absorbance of mixture of Basellamucilage and MeOH. IC50 was calculated as the finalconcentration of Basella mucilage to reduce theabsorbance of DPPH by 50%.

9. Tyrosinase inhibitor assay By using method of Chan et al., 2008, Basella

mucilage was dissolved in 0.1 M sodium phosphatebuffer pH 6.8 into concentrations of 0.125, 0.25,0.5, 1, 2 mg/ml, respectively. 80-L of sample wasadded to 40 L of 100 units/mL tyrosinase in96-well plate. After 10 min, 80 l of 12 mML-DOPA was added, and incubated at 37 oC for 10min, protected from light. The reaction mixtures weremeasured for UV absorbance at 490 nm, andcalculated for % inhibition of each sample. Ascorbicacid was used as positive control.

% radical scavenging = (ADPPH - (Asample- Ablank)) X 100

ADPPH

% inhibition = (Acontrol - (Asample- Ablank)) X 100

Acontrol

When Acomtrol is absorbance of mixture oftyrosinase and L-DOPA; Asample is absorbance ofmixture of Basella mucilage, tyrosinase, and L-DOPA;A blank is absorbance of mixture of Basella mucilage,and tyrosinase. IC50 was calculated as the finalconcentration of the tested sample that reduces theabsorbance of the reaction mixture by 50%.

10. Cell viability test by MTT assayLiver cells (Chang liver cells) were cultured in

96 well plate with 135 l DMEM supplemented with10% fetal bovine serum, penicillin, and streptomycin,at 37 oC in the CO2 incubator (5% CO2) prior to add15 l Basella mucilage in phosphate saline buffer(PBS) to the final concentrations from 0-2 mg/ml.The culture was then incubated for 24 hr. Thepositive control was 0.01 %v/v H2O2, and negativecontrol was PBS. After the culture was washed withPBS, 100 l 5 mg/mL MTT was added and


incubated at 37 oC for 1 hr. The formazan crystal wasredissolved with 150 l DMSO, and measured forUV absorbance at 550 nm.

11. Gel formulation from Basella mucilage andstability test

Gel was formulated into 3 formulae: formulaA, B, and C which contained 0.05 , 0.1 and 0.15 %w/w Basella mucilage, respectively. The gel basecomposed of 0.9% w/w Carbopol 934, 4% v/w 1NNaOH, and 15% w/w propylene glycol. The finishedproducts were tested for stabil i ty byusing heating and cooling cycles method at 4 oC and50 oC cycle, 12 hr each for 6 cycles. The physicalappearances before and after stability test such astexture, color, flavor, pH, and viscosity were recorded.

Results1. Physical properties of Basella mucilageLyophilized powder of Basella mucilage was in

brown-green color with characteristic odor andhygroscopic. It could be swollen in water with theswelling capacity of 4.33 0.29 mL/g dry weight.The redissolved Basella mucilage in water at 1-4%w/w yielded the viscous solution with pH 5.3-5.4.The higher mucilage concentration showed the lowerpH, but increasing of viscosity (Table 1) and moregreen brown color intensity. Basella mucilage formedbrown fragile film after it had been incubated at50 oC for 24 hr. When exposed to the air, the filmbecame sticky, flexible, and stretchable (Fig. 1).

2. Partial purification and monomeric sugardetermination of Basella polysaccharide

The white precipitate of polysaccharide fromBasella mucilage was obtained by EtOH precipitation.After the purification by using DEAE 650M, polysaccharide 11.7 mg (2.6% yield from 450 mg Basellamucilage dry weight) was obtained from 0.5 M NaClelution (Fig. 2). TLC of the acid-hydrolysis polysaccharide clearly indicated that the major sugar wasD-galactose (Fig. 3) with other sugars such asL-arabinose (Fig. 3B, 3C).

1

*+++ = the most intense dark green color, ++ =moderate intense color, + = the less intense color(n = 1)

Table 1 Physical properties of Basella mucilageAppearance Concentration (%w/w)

4 2pH 5.3 5.3 5.4

Viscosity 195.67 143.33 132.33(centipoises)Color intensity* +++ ++ +

Figure 1 Basella mucilage film

Figure 2 Anion chromatography profile of Basellapolysaccharide purification.(DEAE 650M column; UV absorbance at 490nm for sugar detection by using phenol-sulfuricmethod; UV absorbance at 280 nm for proteindetection)


3. Antioxidant and tyrosinase inhibitory activi-ties of Basella mucilage DPPH radical-scavenging assay of Basellamucilage exhibited IC50 at 514.41 g/mL comparedto that of ascorbic acid (2.17 g/mL). However,Basella mucilage showed no inhibitory effect toenzyme tyrosinase when using ascorbic acid aspositive control (IC50 of ascorbic acid was 120 g/mL).

4. Cell viability effect of Basella mucilageBasella mucilage showed mild toxicity to Chang

liver cell as shown in Fig. 4. The mucilage ofconcentration 2 mg/ml exhibited 84.4% cell viability.The estimate IC50 at 5.16 mg/mL could be obtainedfrom the equation y = -10.904x + 106.23 of thelinearity range.

Figure 3 TLC Chromatogram of authentic monosachharides and acid-hydrolyse Basella polysaccharideA. mobile phase CH3CN:H2O (8.5:1.5); B. Mobile phase CH3CN:EtOH (8:2), C. Mobile phase CH3CN:EtOH(13:2). Detection by spraying with 10 % aq. H2SO4 and heat. Dotted circles located the faint spots ofL-arabinose.(lane 1: L-rhamnose; lane 2: D-glucose; lane 3: D-fructose; lane 4: D-sorbitol; lane 5: D-mannose; lane 6:D-xylose; lane 7: D-raffinose; lane 8: L-arabinose; lane 9: D-galactose; lane 10. acid-hydrolyse Basellapolysaccharide fraction from DEAE650M column

Figure 4 Percent viability of Chang liver cell by MTT assay after exposing to various concentration of Basellamucilage. The estimated IC50 was 5.16 mg/mL


5. Gel formulation and stability testAs shown in Table 2, gel formulations from

Basella mucilage were physical stable. No phaseseparation, no precipitation, and no changing of color,

odor, or pH were observed after stability test byheating and cooling cycles method. The viscosities ofgels were 2-4% decreased.

Table 2 Physical properties of Basella mucilage gel preparation before and after the heating and cooling cyclestest

Gel Clear, Clear, Clear, Clear, Clear, Clear,appearance homogenous homogenous homogenous homogenous homogenous homogenous

Flavor normal normal normal normal normal normalColora + + ++ ++ +++ +++

pH 5.76 5.77 5.61 5.59 5.65 5.65Relative 100%b 98% 100%c 96% 100%d 99%viscosity

(%)

Properties Physical properties of Basella gel preparationFormula A

0.05% w/wFormula B

0.10% w/wFormula C

0.15% w/wBefore After Before After Before After

a +++ = the most intense dark green color, ++ = moderate intense color, + = the less intense colorb 20.7 kilopoisesc 20.8 kilopoisesd 19.0 kilopoises

DiscussionThe stem part of Basella alba was selected for

mucilage extraction as it provided highly viscousmaterial when crushing by hand indicated that itcontained more mucilage than leaves. The partialpurification of Basella mucilage composed of only 2.6%polysaccharide because of high water content of thisplant (92%, Maisuthisakul et al, 2007; 2008). DriedBasella mucilage swells in water and gel formedbecause of its viscous characteristic and water-hold-ing capacity. Its mucilage exhibited mild DPPHscavenging activity as from the Basella polysaccharide reported by Palanuvej 2009. From ourphytochemical screening, the aqueous extract of

Basella gave negative result for phenolic compoundassay, therefore it was possible to possess notyrosinase inhibitory activity. For the purificationprocess, protein, and polysaccharide could becoprecipitated by ethanol precipitation, whilechlorophyll was soluble in ethanol, so the whiteprecipitation was obtained and the chromatogram(Fig. 2) showed the mixing of protein andpolysaccharide in the same fractions. Three solventsystems of TLC were used to confirm for sugarcomponent identification. We found that D-galactosewas the major monosaccharide in the acid-hydro-lyzed polysaccharide. The minor amount of L-arabi-nose was also clearly detected although it could not


major compound. The cell toxicity to Chang liver cellshowed tendency of mild toxicity. The gel preparationof Basella mucilage provided good stability that servedfor further development as cosmetic and medicine forskin diseases.

AcknowledgementThe authors wish to thank to Faculty of

Pharmaceutical Sciences, Khon-Kaen University forpartial funding.

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JURNAL DYE Pewarna - Basella Alba Fruit - 2