Biocatalysis and Agricultural Biotechnology 17 (2019) 465–469

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Biocatalysis and Agricultural Biotechnology

journal homepage: www.elsevier.com/locate/bab

Comparison of in vivo antidiabetes activity of snake fruit Kombucha, black T

tea Kombucha and metformin
Elok Zubaidaha, , Chairul Anam Afganib, Umi Kalsumb, Ignatius Sriantac, Philippe J. Blancd
⁎

a
Department of Food Science and Technology, Faculty of Agricultural Technology, Brawijaya University, Jalan Veteran Malang, 65145, Indonesia
b
Department of Pharmacology, Faculty of Medicine, Brawijaya University, Jalan Veteran Malang, 65145, Indonesia
c
Department of Food Technology, Faculty of Agricultural Technology, Widya Mandala Catholic University Surabaya, Jalan Dinoyo 42-44, Surabaya 60265, Indonesia
d
Université de Toulouse, INSA, LISBP, CNRS, UMR5504, INRA, UMR792, Ingénierie des Systèmes Biologiques et des Procédés, 135 Avenue de Rangueil, F-31077
Toulouse, France

ARTICLE INFO ABSTRACT

Keywords: The research compared antidiabetic activity of snake fruit Kombucha, black tea Kombucha and metformin in
Diabetes Mellitus streptozotocin-induced diabetic rats. Snake fruit Kombucha, black tea Kombucha and metformin were orally
Fermentation administered to the diabetic rats daily during a 28-day experiment. Fasting plasma glucose (FPG) levels, su-
Kombucha peroxide dismutase (SOD) activities, malondialdehyde (MDA) levels and lipid profiles (total triglyceride, total
Pancreatic β-cells
cholesterol, LDL-cholesterol and HDL-cholesterol) of the blood plasma were investigated. Pancreas im-
Fasting plasma glucose
munohistochemical study and β-cells quantification were also conducted. The products significantly (p < 0.05)
reduced fasting plasma glucose levels (67–76%) and improved oxidative stress indices and lipid profiles. From
immunohistochemical staining of pancreatic tissues, pancreatic β-cells were also improved in the diabetic rats by
the products. The snake fruit Kombucha compared with the metformin but better than the black tea Kombucha
as a diabetes therapy agent.

1. Introduction studies from our laboratory have demonstrated antioxidant and anti-
diabetic properties of Kombucha from snake fruits (Zubaidah et al.,
Diabetes mellitus (DM) is a metabolic disorder characterized by 2018a, 2018b) to stir further research interests on its comparative an-
hyperglycemia with polyphagia, polyuria, more eating and body weight tidiabetic advantages. However, most of these studies were done
drop, and it is known as a silent killer. It is one of the biggest health without comparing the efficacy of the functional foods with diabetes
problem globally (Ogurtsova et al., 2017). Without proper therapeutic drugs, such as metformin. Such is necessary to understand the relative
management, it leads to severe complications and death. However, it is effectiveness of the intervention. Therefore, the objective of this study
widely managed by insulin injection and drugs, but natural functional was to compare snake fruit and black tea Kombucha, and metformin as
foods without negative effects are being investigated as substitutes, and diabetes therapy agents.
Kombucha is becoming an appealing alternative (Zubaidah et al.,
2018a). 2. Materials and methods
Kombucha is processed by fermentation with symbiotic associations
of bacteria and yeast (Kombucha consortium), it is generally made from 2.1. Materials
black tea and has bioactive compounds with antidiabetic activity by
inhibiting α-amylase activity and suppressing blood glucose levels Snake fruit (Salak Suwaru cultivar) of commercial maturity, com-
(Dufresne and Farnworth, 2000; Goh et al., 2012; Bhattacharya et al., mercial Kombucha starter, black tea, and cane sugar were obtained
2013; Srihari et al., 2013; Jayabalan et al., 2014). Those positive health from plantations, distributors and supermarkets in Malang, East Java,
properties have led to research on other substrates (e.g. snake fruit) for Indonesia as described before (Zubaidah et al., 2018a, 2018b). The
its manufacture. Snake fruit is a tropical fruit that contains vitamins, Salak Suwaru cultivar of the snake fruit was chosen because it showed
minerals, dietary fiber, and bioactive compounds with antioxidant ac- the best characteristics out of the 4 cutivars (Salak Suwaru, Salak
tivities (Aralas et al., 2009; Suica-Bunghez et al., 2016). Previous Doyong, Salak Pondoh and Salak Segaran) we had studied to date

⁎
Corresponding author.
E-mail addresses: [email protected], [email protected] (E. Zubaidah).

https://doi.org/10.1016/j.bcab.2018.12.026
Received 29 June 2018; Received in revised form 4 November 2018; Accepted 26 December 2018
Available online 26 December 2018
1878-8181/ © 2019 Elsevier Ltd. All rights reserved.
E. Zubaidah et al. Biocatalysis and Agricultural Biotechnology 17 (2019) 465–469

(Zubaidah et al., 2018a, 2018b). Metformin was obtained from Phar- due to the higher natural organic acids in snake fruits, while the dif-
macology Laboratory, Faculty of Medicine, Brawijaya University. ferences in total phenolic content, tannin content and antioxidant ac-
tivity might be related to the differences in the raw materials; the su-
2.2. Kombucha preparation and analysis gared black tea extract and the sugared snake fruit juice.

The snake fruit was juiced and its Kombucha was prepared fol- 3.1. Fasting plasma glucose (FPG) level
lowing the procedure as previously described (Zubaidah et al., 2018a).
Black tea Kombucha was prepared according to Ardheniati et al. (2009) Changes in the FPG levels before and after the treatment are pre-
by extracting 10 g of the black tea in 500 mL of boiling water for 10 min sented in Fig. 1. The FPG levels among the DM rat groups (P1, P2, P3,
prior to filtering and sweetening (10% sugar). The sugared tea extract and P4) on day 0 are not significantly different (p > 0.05), while there
was put into a glass jar, cooled to room temperature before adding 10% are significant differences (p < 0.05) with the normal rats (P0). At the
Kombucha starter aseptically. The jar was then covered with a sterile end of the 28-day treatment, the normal and DM rats appeared to show
cheese cloth and incubated at room temperature for 14 days. The constant FPG levels, whereas the FPG levels of the DM groups P2, P3
physicochemical and antioxidant properties of the Kombucha were and P4 decreased. The DM group with the KS (P3) and metformin (P4)
analyzed as before (Zubaidah et al., 2018a). administration showed lower FPG levels than the DM group with the KT
(P2).
2.3. Animal experiment and analysis Metformin is an antidiabetic drug, and it works by increasing the
sensitivity of the liver and peripheral tissues to insulin without affecting
Twenty five healthy three-months old male Wistar rats were divided insulin secretion and increasing glucose uptake in the peripheral tissues
randomly into 5 groups with essentially 5 replicates - group 1 (P0), to reduce insulin resistance (Iida et al., 2003). The snake fruit Kom-
normal; group 2, diabetic, DM (P1); group 3, DM with the black tea bucha decreased the level of FPG, because of its high content of anti-
Kombucha (KT) at a dose of 5 mL/kg BW/day (P2); group 4, DM with oxidant compounds such as phenolic, tannin and other bioactives, as
the snake fruit Kombucha (KS) at a dose of 5 mL/kg BW/day (P3); and well as some other organic acids such as citric, lactic, butyric, and
group 5, DM with the metformin at the recommended dose of 45 mg/kg propionic acids (Ostman et al., 2005; Zubaidah et al., 2018a). Anti-
BW/day (P4). The DM rats were induced with streptozotocin (Nacalai oxidant compounds decrease FPG levels through increasing cellular
Tesque, Japan) intraperitoneally at a dose of 45 mg/kg BW. The rats glucose uptake. Increased insulin secretion will have implications for
accessed standard diet and water ad libitum during the 28-day ex- the body's ability to utilize blood glucose for normal metabolism. Gly-
periment, and groups 3–5 were respectively administered with tea colysis, glycogenesis and lipogenesis are some of the metabolisms of
Kombucha, snake fruit Kombucha and metformin on a regular basis insulin-regulated glucose (Dufresne and Farnworth, 2000; Aloulou
once a day. et al., 2012). Antioxidant compounds can also inhibit glucose absorp-
Fasting plasma glucose (FPG) level measurements were conducted tion in the small intestine by decreasing glucose-conducting activity,
on days 0 and 28, and at the end of the experiment, the rats were sa- such as sodium-glucose transport protein 1 (GLUT1), glucose trans-
crificed by cervical dislocation. The blood was used to analyse for su- porter 5 (GLUT 5) and glucose transporter 2 (GLUT 2). The gross effect
peroxide dismutase (SOD) activity, malondialdehyde (MDA) and lipid of these is to reduce the glucose that enters the bloodstream (Kwon
profiles (total cholesterol, triglycerides, high-density lipoprotein (HDL) et al., 2007). Consequently, it can be inferred that the snake fruit
cholesterol, and low-density lipoprotein (LDL) cholesterol levels) as Kombucha had a comparable effect as the metformin and a better effect
before (Zubaidah et al., 2018b). on FPG than the black tea Kombucha.

2.4. Immunohistochemical staining (IHC) 3.2. Superoxide dismutase (SOD) activity and malondialdehyde (MDA)
levels
After sacrifice, the rat pancreas was taken and fixed in 10% buffered
formalin for 24 h, and slides were made by standard methods using The SOD activity and MDA levels reflect the body oxidation status
paraffin. IHC staining was done as in Beesley (1995), and it involved (Bhattacharya et al., 2013), and Table 2 shows that the rats in the DM
treating with diamino benzidine (DAB) for 3 min and counter staining group had higher oxidative stresses than the rats in the normal group,
with mayers haematoxilin for 3 min. Insulin was visualized as brown with the Kombucha and metformin administered rats having higher
color. Quantification of β-cells was conducted according to Suarsana SOD activities and lower MDA levels than the DM group to indicate
et al. (2010) by calculating the average of β-cells. improved oxidation status in the DM rats. This is expected as the an-
tioxidant compounds, polyphenols (Table 1) in the Kombucha would
2.5. Statistical analysis have contributed to this desirable outcome. The potent antioxidant
ability of phenolic compounds is thought to be due to the ability to
The data were analyzed by analysis of variance (ANOVA), followed donate electrons or hydrogen atoms from the -OH group, which makes
by the LSD test at p < 0.05. them unstable but the resonance of electrons by the benzene ring sta-
bilizes them for their desirable properties (Scalbert et al., 2005). This
3. Results and discussion enhances Kombucha contributions to the body antioxidant enzyme
system thereby reducing the adverse effects of diabetes mellitus (Zhang
Kombucha fermentation is a biochemical process, whereby the and Tsao, 2016). It is noteworthy that the Kombuchas were either
Kombucha consortium transforms substrates into products such as comparable or better than the metformin in these body oxidation in-
acetic acid and others. Table 1 shows the characteristics of the Kom- dices (Table 2).
bucha, and the differences before and after the fermentation are ex-
pected from the microbial activities of the Kombucha consortium on 3.3. Lipid profiles
sucrose, which is the main carbon source (Malbasa et al., 2011; Jasman
and Widianto, 2012). Both Kombucha decreased in pH, total sugar and Table 3 shows the total cholesterol, total triglyceride and LDL-c of
total solids and increased in total acidity, total phenolic content, tannin the diabetic rat groups were higher than those of the normal rat group,
content, and total antioxidant activity; consistent with previous studies due to lipid metabolism disorders. Administering the Kombuchas and
(Kallel et al., 2012; Johnson and De Mejia, 2016). The total acidity in metformin to the diabetic rat groups improved the lipid profiles, and
the snake fruit Kombucha was higher than that in the black tea, possibly the snake fruit Kombucha was more effective in the improvement than

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Table 1
Chemical characteristics of the black tea Kombucha and snake fruit Kombucha#.
Parameter Kombucha Type

Black tea Kombucha Snake fruit Kombucha

Day 0 Day 14 Day 0 Day 14

Total acidity (%) 0.09 ± 0.04 0.42 ± 0.07 * *a 0.57 ± 0.14 1.56 ± 0.17 * *b
pH 4.77 ± 0.26 3.08 ± 0.10 * *a 3.91 ± 0.19 3.22 ± 0.09 * *a
Total sugar (%) 9.06 ± 1.01 6.78 ± 0.06 * *a 10.50 ± 0.44 7.76 ± 0.03 * *a
Total solid (%) 9.90 ± 0.10 8.13 ± 0.06 * *a 13.93 ± 0.06 12.88 ± 0.08 * *b
Total phenolic content (mg/L GAE) 228.15 ± 24.16 400.06 ± 47.99 * *a 281.01 ± 11.28 535.59 ± 1.96 * *b
Tannin (mg/L TAE) 530.89 ± 82.51 704.81 ± 32.25 * *a 496.67 ± 7.64 619.00 ± 39.15 * *b
Antioxidant activity/DPPH (%) 79.82 ± 4.94 89.33 ± 1.25 * *a 86.38 ± 1.18 91.73 ± 3.64 * *b

#
Values are means ± standard deviations (n = 3). GAE = Gallic acid equivalent, TAE = Tannic acid equivalent, DPPH = 2,2-diphenyl-1-picrylhydrazyl.
For each parameter, values with symbol* * indicate significant differences between days 0 and 14 on each observation parameter (p < 0.05). Values with same
lettera,b indicate no significance differences between snake fruit Kombucha and black tea Kombucha on each parameter (p > 0.05).

is through donating hydrogen atoms to lipid radicals (R*, ROO*) and

converting them to a more stable form and slowing the auto-oxidation
rate. The addition of a low concentration of primary antioxidants to
lipids reportedly inhibits or prevents oxidation of fatty acids (Gordon,
1990; Taku et al., 2007). Phenolic compounds can also increase HDL-c
and lower total cholesterol, triglycerides, and LDL-c by aiding meta-
bolisms in generating energy, contributing to fat metabolisms, in-
creasing bile acid excretions, decreasing total cholesterol absorptions
by binding cholesterol carriers as they pass through the brush border
membranes. This consequently decreases the production of lipoproteins
and increases the activity of lecithin cholesterol acyl transferase
(LCAT). LCAT is an enzyme that converts free cholesterol to cholesterol
esters and plays a role in HDL-c metabolisms (Carvajall-Zarrabal et al.,
2005; Zern and Fernandez, 2005; Tan et al., 2007; Rahimi-Madiseh
Fig. 1. Effect of the Kombucha and metformin administration on indices of the et al., 2017).
fasting plasma glucose (FPG) levels in the rats. Values are means ± SD (n = 4).
Bars with the same letters are not significantly different (p > 0.05). Lower case
3.4. Pancreas immunohistochemistry (IHC) study
letters are for day 0, while capital letters are for day 28.

The result of IHC staining is shown in Figs. 2 and 3. The results

Table 2 showed increasing Langerhans island structures and insulin secretions
Effect of the black tea Kombucha, snake fruit Kombucha and metformin ad- in the three treated (Kombucha and metformin) groups (Fig. 3). The
ministration on SOD activity and MDA level in rats. size and shape of the structures from the DM group were irregular and
Treatment SOD (unit/100 µL) MDA (ng/100 µL) smaller than those of the normal group (P0) and the three groups of KT,
KS and metformin. In addition, the DM group showed a very low im-
a
P0 (Normal) 52.47 ± 2.23 0.28 ± 0.03d munoreactive response (brown color) to anti-insulin, which indicated
P1 (DM) 17.66 ± 4.79d 0.83 ± 0.02a
P2 (DM + KT) 39.50 ± 11.71b 0.44 ± 0.02bc
low levels of insulin production. The DM rat groups with the Kombucha
P3 (DM + KS) 44.55 ± 5.98b 0.46 ± 0.02bc and metformin improved in the Langerhans island structures, and in the
P4(DM + Metformin) 31.78 ± 3.79c 0.39 ± 0.02c size, shape, distributions, and numbers of the β-cells, as well as the high
intensity of the brown color when compared with the DM rats (Fig. 3).
DM: Diabetes Mellitus, KT: Black tea Kombucha KS: Snake fruit Kombucha. The DM group with metformin (P4) had a high number of the
Values in a column with the same letters are not significantly (p > 0.05) dif-
pancreatic β-cells, which is not significantly (p > 0.05) different from
ferent.
the snake fruit Kombucha. Metformin has adenosine monophosphate-
This applies to all tables, where they appear.
activated protein kinase (AMPK) enzyme that plays a role in repairing
HbA1c, the main parameter of blood glucose. In addition, it can reduce
the black tea Kombucha, possibly due to its higher bioactive com-
hepatic glucose production, lower LDL and triglyceride levels, increase
pounds (Table 1) as discussed above. The snake fruit Kombucha also
HDL levels, decrease platelet aggregation, increase fibrinolytic activity
significantly showed better effects than the metformin (Table 3).
and improve weight, reduce the risk of hypoglycemia, and increase
The mechanism of phenolic compounds in improving lipid profiles
insulin sensitivity with a concomitant improvement of pancreatic

Table 3
Effect of the black tea Kombucha, snake fruit Kombucha and metformin administration on the lipid profile serum levels inthe rats.
Treatment Cholesterol (mg/dL) Triglycerides (mg/dL) HDL (mg/dL) LDL (mg/dL)

P0 (Normal) 44.75 ± 11.22 b 48.00 ± 21.12c 66.75 ± 1.71 a 6.75 ± 6.29c

P1 (DM) 75.25 ± 10.50 a 102.75 ± 22.94 a 37.75 ± 5.70c 14.25 ± 3.40 a
P2 (DM+KT) 51.50 ± 5.20c 73.25 ± 24.53 b 40.50 ± 9.95 b 11.50 ± 1.91 b
P3 (DM+KS) 44.75 ± 3.79 b 52.50 ± 28,84c 58.75 ± 2.87 a 7.25 ± 1.26c
P4 (DM+Metformin) 52.50 ± 9.00c 91.75 ± 26.71 b 56.75 ± 21.65 a 11.00 ± 0.82 b

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Fig. 2. Effect of the Kombucha and metformin administration on the pancreatic cells in rats evaluated by the IHC staining (400× magnification). PL: Langerhans
Island, EKS: Exocrine glands (acini). Yellow arrow: Pancreatic β-cells, which have immunoreactivity to anti-insulin. Green arrow: Endocrine cells, which are not
immunoreactive to anti-insulin. Red arrow: Empty space by necrosis, DM: Diabetes Mellitus, KT: black tea Kombucha, KS: snake fruit Kombucha, BW: Body Weight.
(For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article).

bioactive compounds of the snake fruit Kombucha (Table 1) can also act
as insulin promoters and secretagoues. Insulin promoters are involved
in the development of pancreas that aids maturation of β-cells, whereas
insulin secretagoues stimulate insulin secretions from pancreatic β-
cells. Phenolics and tannins have also been shown in in vitro studies to
increase insulin secretions from pancreatic β-cells, and this synergizes
with antioxidant compounds in increasing insulin secretions. The rat
groups administered with the snake fruit Kombucha and metformin
showed the best results and that the snake fruit Kombucha can be an
effective diabetes therapy agent.

4. Conclusions

The three treatments, the snake fruit Kombucha, black tea

Kombucha and metformin, were effective as diabetes therapy agents in
Fig. 3. Effect of the Kombucha and metformin administration on the number of the STZ-induced rat model by lowering FPG, improving oxidation stress
pancreatic β-cells. Values are mean ± SD of β = cells of five Langerhans is- statuses and lipid profiles. Improvements of pancreas were evident in
lands. The same letter indicates no significant difference (p > 0.05). DM: the higher Langerhans islands and the β-cells numbers by these three
Diabetes Mellitus. treatments. However, the snake fruit Kombucha was as effective as the
metformin in managing the induced diabetes, and more than the black
performances (Gunton et al., 2003). The DM group with the snake fruit tea Kombucha. Hence, snake fruit Kombucha can potentially substitute
Kombucha treatment had the pancreatic β-cells higher than those of the metformin as a diabetes therapy agent, and this paves way for an ex-
black tea Kombucha, possibly due to its higher antioxidant compounds tensive human trial to ascertain this.
as discussed above that can protect and repair the pancreatic β-cells and
increase insulin secretions (Zubaidah et al., 2018a, 2018b). The

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