2.2 %mg(w/w)

97.8 %mg(w/w)

Comparison of Different Methods and Storage Conditions for Anthocyanin Extraction from Black Goji Berry

(Lycium ruthenicum)

Supamas Kruawan1, Vichulada Janchom2 and Apinun Kanpiengjai1*

1Division of Biochemistry and Biochemical Technology, Department of Chemistry, Faculty of Science,

Chiang Mai University, Chiang Mai 50200, Thailand. 2Detox (Thailand) Company Limited

[1] Dhar, P. Tayade, A., Ballabh B., Chaurasia, O.P., Bhatt, R.P., and Srivastava, R.B. (2011). Lycium ruthenicum Murray: A less-explored but high-value medicinal plant from Trans-Himalayan cold deserts of Ladakh, India. Plant

Archieves. 11(2): 583-586.

[2] Martin J., Navas M.J., Jimenez-Moreno A.M., and Asuero A.G. Anthocyanin pigments: Importance, sample preparation and extraction. In: Soto-Hernandez M. (ed). Phenolic compounds-Natural sources, importance and

applications. IntechOpen; 2017: p.117-152.

[3] Islam T., Yu X., Badwal T. S., and Xu B. (2017). Comparative studies on phenolic profiles, antioxidant capacities and carotenoid contents of red goji berry (Lycium barbarum) and black goji berry (Lycium ruthenicum). Chemistry

Central Journal, 11(1): 59.

[4] Kupina S., Fields C., Roman, M. C., and Brunelle, S. L. (2019). Determination of Total Phenolic Content Using the Folin-C Assay: Single-Laboratory Validation, First Action 2017.13. Journal of AOAC International, 102(1), 320-321.

[5] Lee J., Durst R., and Wrolstad R. (2005). AOAC official method 2005.02: total monomeric anthocyanin pigment content of fruit juices, beverages, natural colorants, and wines by the pH differential method.

Official methods of analysis of AOAC International, 37, 37.

Acknowledgements➢ Advisor : Dr. Apinun Kanpiengjai

➢ Ms. Vichulada Janchom

➢ Division of Biochemistry and Biochemical Technology, Department of Chemistry,

Faculty of Science, Chiang Mai University

➢ Detox (Thailand ) company Limited.

ABSTRACT

Black goji berry is one of the most popular berries that is widely used in traditional Chinese medicine. It contains a water-soluble pigment, anthocyanin, as the major bioactive

compounds, thus a natural color enhancer and health benefits. The objectives of this study were to determine different methods for extraction of anthocyanin from black goji berry and

evaluate storage stability of anthocyanin. A total of three different extraction solvents including water, 60% ethanol and the mixture of ethanol-acetic acid-water in the ratio of 50:2:48

(v/v/v) were applied to the black goji berry and the extraction was performed at 30ºC and 50ºC. The extracted anthocyanin content was determined by the spectrophotometric pH

differential method. The results revealed that monomeric anthocyanin content of 180.74±20.9 mg/100 g black goji berry was obtained when the mixture of ethanol-acetic acid-water

was applied with the extraction temperature at 50ºC. This amount was of higher than those extracted by other conditions. Moreover, storage of the anthocyanin extracted by this

condition at -20ºC for 24 h resulted in 85% residual anthocyanin content. With regard to the extraction by 60% ethanol at 50ºC, the anthocyanin content of 151.88±12.5 mg/100 g

black goji was obtained, and this amount was retained with 100% residual anthocyanin content after the storage at both 4 and -20ºC for 24 h. Therefore, this extraction method and

storage condition is considered an appropriate protocol for production of anthocyanin from black goji berry.

Lycium ruthenicum

Lycium ruthenicum or black goji

berry is an important medicinal plant. It is

also known as black wolfberry that is native

of the Himalayan Mountains in Central Asia

where it is revered for its health benefits.

Black Goji fruits are a rich source of

carotenoids , anthocyanins , vitamins A, C,

B1 and B2 ; trace mineral like iron, zinc,

selenium and alkaloid betaine that helps to

produce choline [1].

Prevention of diabetes

Prevention of cardiovascular

and cerebrovascular diseases

Lowering blood pressure

Anti-inflammatory, anti-bacterial

and antioxidant activities.

Reduction of cancer cell

proliferation [3]

Anthocyanin

Fig.1 Structure of the major

anthocyanins-3-O-glucoside

❖ Anthocyanins are the largest

group of phenolic pigments and

the most important group of water-

soluble pigments in goji berry [2].

❖ Among the anthocyanin pigments,

cyanidin-3-glucoside is the major

anthocyanin found in most of the

plants.

References

Goji berry sample

3 g of dry ground goji berry

Extracted twice with 30 mL of different

extraction solvents

Placed in 30 ºC

water bath for 24 h

7,500 rpm for 5 min.

Collected supernatant

- Total phenolic content (TPC) [4]

- Monomeric anthocyanin content [5]

Ground with pestle and mortar

Storage at 4˚C in

dark condition

Storage at -20˚C in

dark condition

Extraction with

60%ethanol

Extraction with ethanol/

acetic acid / water

(50:2:48)

Extraction

with water

1 2 3

Solvent

The mixture of ethanol-acetic acid-water (50:2:48 v/v/v) in combination with

extraction temperature of 50ºC was the best anthocyanin extraction condition.

The best conditions for storage of anthocyanin was in 60% ethanol and

stored at 4 and -20ºC for 24 h.

CONCLUSIONS

Fig. 2 Anthocyanin content of black goji berries after extraction at 30ºC

and 50ºC with water, 60%ethanol and ethanol-acetic acid-water

Fig. 3 Effect of temperature on stability of

anthocyanins extracted from various organic

solvents at 30ºC

EXTRACTION

Fig. 4 Effect of temperature on stability of

anthocyanins extracted from various organic

solvents at 50ºC

Anthocyanin

Other phenoliccompounds

STORAGE CONDITIONS

Anthocyanin content

in Black Goji berry

Preparation

Placed in 50 ºC

water bath for 24 h

% Residual anthocyanin

Fig. 5 Anthocyanin obtained after

extracted the black goji berry by

ethanol-acetic acid-water (50:2:48) at 50ºC

0

50

100

150

200

30 ºC 50 ºC

Anth

ocyanin

conte

nt(

%m

g)

Temperature (ºC)

Water 60%Ethanol Ethanol : Acetic acid : Water

0

40

80

120

160

4 ºC - 20 ºC

%R

esid

ual A

nth

ocyanin

Temperature (ºC)

Water 60%Ethanol Ethanol : Acetic acid : Water

0

40

80

120

160

4 ºC - 20 ºC

%R

esid

ual A

nth

ocyanin

Temperature (ºC)

Water 60%Ethanol Ethanol : Acetic acid : Water

INTRODUCTION RESULTS

METHODOLOGY

Spectrophotometric pH differential method