Study of Tinospora cordifolia stem and Senna siamea leaf and pods: An in vitro therapeutic approach for leukemia

Abstract

BACKGROUND

Tinospora cordifolia (T. cordifolia) Miers is an evergreen and dioecious herb of the Menispermaceae family. Senna siamea (S. siamea) Lam. is a medium-sized tree of the Fabaceae family, and it is known for its nutritional, economic, and medicinal importance.

AIM

To analyze the phytochemicals and enzymes in T. cordifolia and S. siamea and perform an in vitro therapeutic assay.

METHODS

The phytochemical compounds in T. cordifolia stem aqueous extract (TCAE), S. siamea leaf aqueous extract (SLAE), and S. siamea pod aqueous extract (SPAE) were estimated. Activity of catalase, superoxide dismutase, peroxidase, polyphenol oxidase, and ascorbate oxidase was quantified in protein extracts from T. cordifolia stem, S. siamea leaf, and S. siamea pods, and an in vitro therapeutic assay was performed in THP-1 cells.

RESULTS

Quantitative analysis revealed that the total flavonoid content was high in SLAE. The total phenolic compound content was high in SLAE. The total tannin content was high in SPAE. The enzymatic activity of catalase, peroxidase, and ascorbate oxidase was high in T. cordifolia stem, and the enzymatic activity of superoxide dismutase and polyphenol oxidase was high in S. siamea pods. TCAE, SLAE, and SPAE induced THP-1 cell apoptosis due to an increase in reactive oxygen species and a decrease in mitochondrial membrane potential activity.

CONCLUSION

T. cordifolia and S. siamea may have therapeutic potential in leukemia, but further clinical studies are needed.

Key Words: Tinospora cordifolia; Senna siamea; Quantitative analysis; Enzymatic activity; Therapeutic approach

Core Tip: The following study focused on defining the therapeutic properties of aqueous extracts of Tinospora cordifolia stem and Senna siamea pods and leaf in a leukemia cell line. Both plants had therapeutic properties and may be potential treatments for myeloid leukemia, as they induced apoptosis through an increase in reactive oxygen species activity and a reduction in mitochondrial membrane potential. The following study was performed in in vitro and needs further in vivo and clinical studies to confirm our observations.

INTRODUCTION

Tinospora cordifolia (T. cordifolia) Miers (Guduchi) is an evergreen climber herb of the Menispermaceae family. In the Indian system of medicine, this plant has significant importance, as all parts of the plant are medicinally used. Its stem has a higher alkaloid content than its leaves[1]. Locally, it is also known as Giloy or Guduchi and has been documented in ayurvedic writings to possess antidiabetic and immunomodulatory properties[2]. T. cordifolia is well known as an Indian bitter and is prescribed for fevers, diabetes, dyspepsia, jaundice, urinary problems, skin diseases, chronic diarrhea and dysentery. It is also useful in treating heart disease, leprosy, and helminthiasis[3] and is thought to improve the immune system. Several studies have found that different parts have distinct health benefits[4]. T. cordifolia is thought to have antispasmodic, antipyretic, antineoplastic, hypolipidemic, hypoglycemic, immunopotentiation, and hepatoprotective properties.

Senna siamea (S. siamea) Lam. is a medium-sized common tree of the Fabaceae family found in Southeast Indian regions[5]. It is a tropical plant well known for its nutritional, medicinal, and economic importance, and it is used in the treatment of fever, malaria, diabetes, hypertension, asthma, constipation, diuresis, jaundice, abdominal pain, and menstrual pain. S. siamea is also medicinally important for pathological complications. In some parts of Asia, this plant is used as a vegetable. The major phytochemical compounds in this plant are coumarins, alkaloids, flavonoids, glycosides, triterpenoids, sterols, and other polyphenols[6]. It is an angiosperm native to Southeast Asian countries like Burma, Ceylon, India, Japan, Malaysia, Sri Lanka, and Thailand and is distributed in some parts of Southern Africa, Latin America, and Oceania.

S. siamea is a shrub of 10-12 m tall but can reach up to 20 m. The young bark is grey and smooth, the leaves are alternate, the flowers are bright yellow in color, and the fruits are flat indehiscent pods constricted between the seeds. The number of seeds per pods is 20, they are bean-shaped, greenish-brown in color, and 8-15 mm long. The plant is commonly used in cattle rearing, agriculture, the environment, and furniture and has various medicinal uses[7]. In Sanskrit, it is known as swarn patri and is used for different conditions. This plant is also known as the johar medicinal plant.

One of the compounds present in the leaves is Casiarine A, which has antimalarial activity[8]. S. siameae has dose-dependent cytotoxic effects against Raniceps raninus (R. raninus) and Saccharomyces cerevisiae (S. cerevisiae)[9]. The leaf of S. siamea is rich in potassium, magnesium and phosphorus, and contains phytochemicals like saponins, flavonoids, phenolics, steroids, and alkaloids[10]. Aqueous extract of S. siamea has weak anti-plasmodial activity. Treatment with the diethyl ether extract of S. siamea in Plasmodium berghei suppressed the expression of Pb-EMPI[11]. Petroleum ether extract of S. siamea has good inhibitory activity against α-amylase and α-glucosidase and good binding affinity against the protein 1HNY with the compounds sennamin A, cassismin B, sennarin B, and chrolisiamone A present in S. siamea leaves[12].

In this study, the phytoconstituents and enzymes in the stem of T. cordifolia and S. siamea leaf and pods were analyzed, and an in vitro therapeutic approach was performed to study whether either plant has any anti-leukemic effect on the THP-1 cell line. The objective of the study was to quantitatively analyze the phytoconstituents and enzymatic activity in T. cordifolia stem and S. siamea leaf and pod extracts, and define their therapeutic efficacy in the THP-1 cell line.

MATERIALS AND METHODS

Collection and authentication of plant samples

The plant samples of T. cordifolia stem and S. siamea leaf and pods were collected from the Bharathiar University campus and were authenticated in the Botanical Survey of India, Tamil Nadu Agricultural University, Tamil Nadu, India.

Preparation of extract

Aqueous solvent was chosen for the preparation of the extract using the maceration method. The samples of both the plant T. cordifolia stem and S. siamea leaf and pods were collected and dried. After shade drying, the plant samples were ground into a fine powder. The ground plant samples were used for aqueous extract preparation in a 1:10 ratio with 10 g of powdered sample and 100 mL distilled water and kept overnight for 12 hours at 37 °C. The extract was then kept in a shaker for 24 hours and stored for future use.

After extract preparation, quantitative analysis of the total flavonoid, total phenolic compound content, and total tannin content was performed in all three prepared extracts. The three extracts obtained were T. cordifolia stem aqueous extract (TCAE), S. siamea leaf aqueous extract (SLAE) and S. siamea pods aqueous extract (SPAE).

Quantitative analysis

Total flavonoid estimation: Estimation of the total flavonoid content in TCAE, SLAE, and SPAE was performed using the aluminium-chloride colorimetric method. A total of 100 μL of each plant extract was isolated, and 0.3 mL 5% sodium nitrate was added and mixed well. Then, 0.3 mL 10% aluminum chloride was added and incubated at room temperature for 6 minutes. After incubation, 1 mL of 1 M sodium hydroxide was added, and distilled water was added to a final volume of 10 mL. Readings were acquired at 510 nm in a UV/Vis spectrophotometer. For the flavonoid standard, 200 μg, 400 μg, 600 μg, 800 μg, and 1000 μg quercetin were measured[13].

Total phenolic compound estimation: Estimation of the total phenolic compounds in TCAE, SLAE, and SPAE were performed using the Folin-Ciocalteau method[14]. Briefly, 1.25 mL of 10-fold diluted Folin-Ciocalteau reagent was added to each extract. After a 5-minute incubation, 5 mL of 7.5% sodium carbonate was added to the mixture and incubated at room temperature for 90 minutes in the dark. Each solution was analyzed at 760 nm in a UV/Vis spectrophotometer. Gallic acid was prepared at 200 μg, 400 μg, 600 μg, 800 μg, and 1000 μg as a standard[14].

Total tannin estimation: Total tannins were estimated in TCAE, SLAE, and SPAE using the Folin-Ciocalteau method[15]. Briefly, 0.5 mL of 10-fold diluted Folin-Ciocalteau reagent was added to each extract and mixed well. Then, 35% sodium carbonate was added, mixed, and incubated at room temperature for 30 minutes. Readings were taken at 700 nm in a UV/Vis spectrophotometer. Tannic acid was used as standard (200 μg, 400 μg, 600 μg, 800 μg, and 1000 μg).

Quantitative analysis of enzymatic activity

Catalase activity: To measure catalase activity of the T. cordifolia stem and S. siamea pod and leaf, total protein was isolated by grinding 1 g of plant sample in phosphate buffer solution (PBS) and centrifuging for 15 minutes at 4 °C at 10000 rpm. The supernatant was collected for further processing. The amount of total protein was estimated by Bradford assay. For estimating catalase activity, reaction mixtures were prepared with 50 mmol/L PBS (pH 7.0), 0.1% Triton-X-100, and 10.5 mmol/L H₂O₂. The protein extracts of T. cordifolia stem and S. siamea leaf and pods were added in the dark, and readings were taken in a UV/Vis Spectrophotometer for 2 minutes at 30-second intervals at 240 nm[16].

Superoxide dismutase: Superoxide dismutase (SOD) activity was spectrophotometrically estimated by determining the inhibition of photoreduction using nitro blue tetrazolium. A reaction mixture of 3 mL was prepared by adding 50 mmol/L PBS (pH 7.6), 0.1 mmol/L EDTA, 50 mmol/L Na₂CO₃, 12 mmol/L L-methionine, 50 μM nitro blue tetrazolium, and 10 μM riboflavin. To the above mixture, 100 μL of protein extracts of T. cordifolia stem and S. siamea leaf and pods were added and kept under white light at room temperature for 15 minutes. After incubation, the reaction mixtures were analyzed in a UV/Vis spectrophotometer at 560 nm[17].

Peroxidase: Peroxidase activity was estimated spectrophotometrically based on pyrogallol oxidation in the presence of H₂O₂. A 3-mL assay mixture was prepared containing 0.1 M PBS, pyrogallol, H₂O_2, and the prepared T. cordifolia stem and S. siamea leaf and pods protein extracts (500 µL). The mixtures were analyzed at 420 nm in a UV/Vis spectrophotometer for 3 minutes at 30 seconds intervals[18].

Polyphenol oxidase: Polyphenol oxidase (PPO) activity in T. cordifolia stem and S. siamea leaf and pods was measured by extracting total protein. A reaction mixture of 6 mL was prepared for estimation. The solution consisted of 50 mmol/L PBS (pH 5.5), 100 mmol/L catechol, and the T. cordifolia stem and S. siamea leaf and pods protein extracts (500 μL). The prepared reaction mixtures were analyzed in a UV/Vis spectrophotometer at 420 nm for 2 minutes at an interval for 30 seconds[19].

Ascorbate oxidase: The ascorbate oxidase activity of T. cordifolia stem and S. siamea leaf and pods were estimated by extraction. Protein extracts were added to PBS with ascorbic acid, and the readings were taken at 265 nm in a UV/Vis spectrophotometer every 30 seconds for 2 minutes[20].

Cell culture

The THP-1 myeloid leukemia (ML) cell line was obtained from the National Centre for Cell Science Pune. It was maintained at 37 °C with 5% CO₂. The following study was performed to identify the medicinal effect of TCAE, SLAE, and SPA.

Cell cytotoxicity: Cell cytotoxicity of 50-200 μg TCAE, SLAE, and SPAE was tested using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium (MTT) assay. THP-1 cells were seeded for 24 hours and then incubated with different concentrations of TCAE, SLAE, or SPAE. After incubation, the MTT reagent was added and incubated for 3-4 hours and read at 570 nm in a plate reader[21].

Reactive oxygen species assay: THP-1 cells were seeded and grown until 80% confluence. Cells were washed with PBS, and 2’,7’-dichlorodihydrofluorescein diacetate was incubated at 37 °C for 30 minutes. After incubation, the cells were washed with PBS to remove excess stain. TCAE, SLAE, or SPAE (50-150 μg) was added to the cells and incubated for 1 hour. Readings were taken at 535 nm in a plate reader to estimate the reactive oxygen species (ROS) levels[22].

Mitochondrial membrane potential assay: THP-1 cells were grown in 6-well plates to 80% confluence then treated with TCAE, SLAE, or SPAE (50-150 μg). After washing with PBS to remove any residue, cells were treated with Rho123 and incubated at 37 °C for 30 minutes in the dark. After washing with PBS, readings were taken at 530 nm in a plate reader to estimate mitochondrial membrane potential (MMP)[23].

Cell cycle assay: THP-1 cells were cultured for 24 hours and treated with TCAE, SLAE or SPAE (150 μg) for 24 hours. Cells were harvested and centrifuged at 25 °C at 300 g for 5 minutes. After centrifugation, the supernatant was discarded and the cell pellet was washed twice with PBS before being fixed in 70% ethanol and analyzed using an FC500 Flow Cytometer, Beckman Coulter, United States[21]. A one-way analysis of variance (ANOVA) was performed.

Apoptosis assay: THP-1 cells were cultured in 6-well plates overnight for 24 hours in 5% CO₂ and 37 °C. The cells were then treated with 150 μg TCAE, SLAE or SPAE and incubated for 24 hours. After incubation, the cells were centrifuged and the supernatant was discarded. The cells were resuspended in PBS and washed twice. The cell pellet was resuspended in binding buffer with annexin V, vortexed gently, and incubated for 15 minutes at 4 °C. After incubation, 2 μL potassium iodide was added with binding buffer, vortexed, and then analyzed using a FC500 Flow Cytometer, Beckman Coulter, United States[21].

Statistical analysis

One-way ANOVA statistical tests were peformed using the statistical tool SPSS software version 19 (IBM Corp., Armonk, NY, United States). A P value < 0.05 was considered statistically significant.

RESULTS

TCAE, SLAE and SPAE were prepared and analyzed.

Quantitative analysis

Total flavonoid content: Total flavonoids were high in SLAE (3.76 g/100 mL and 3.71 g/100 mL), followed by SPAE (3.62 g/100 mL and 3.21 g/100 mL) and TCAE (2.68 g/100 mL and 2.30 g/100 mL) (Figure 1A, Figure 2, Table 1).

Figure 1 Standard graph of flavonoids, phenolic compounds and tannin. A: Standard graph for flavonoids of the quercetin of the concentration range 200-1000 μg; B: Standard graph for phenolic compound of gallic acid of the concentration range of 200-1000 μg; C: Standard graph for tannins for tannic acid the concentration range of 200-1000 μg.

Figure 2 Estimation of total flavonoid content. The above graph represents the concentration of total flavonoid compound present in the Tinospora cordifolia stem aqueous extract, Senna siamea leaf aqueous extract and Senna siamea pods aqueous extract. The highest flavonoid is found in Senna siamea leaf aqueous extract of 3.76 g/100 mL. TCAE: Tinospora cordifolia stem aqueous extract; SLAE: Senna siamea leaf aqueous extract; SPAE: Senna siamea pods aqueous extract.

Table 1 Total flavonoid content in the Tinospora cordifolia stem aqueous extract, Senna siamea leaf aqueous extract and Senna siamea pods aqueous extract.

Samples	Round 1 (g/100 mL)	Round 2 (g/100 mL)
TCAE	2.68	2.3
SLAE	3.76	3.71
SPAE	3.62	3.21

In the following table, the total flavonoid concentration of Tinospora cordifolia stem aqueous extract, Senna siamea leaf aqueous extract and Senna siamea pods aqueous extract and control cells are estimated and higher total flavonoid content is present in SLAE with 3.76 g/100 mL. TCAE: Tinospora cordifolia stem aqueous extract; SLAE: Senna siamea leaf aqueous extract; SPAE: Senna siamea pods aqueous extract.

Total phenolic compound content: Total phenolic compounds were highest in TCAE (2.06 g/100 mL and 2 g/100 mL), followed by SLAE (1.39 g/100 mL and 1.10 g/100 mL) and SPAE (1.96 g/100 mL and 1.87 g/100 mL). (Figure 1B, Figure 3, Table 2).

Figure 3 Estimation of total phenolic compound content. The above graph represents the concentration of total phenolic content in the Tinospora cordifolia stem aqueous extract, Senna siamea leaf aqueous extract and Senna siamea pods aqueous extract. The highest phenolic compound is found in Tinospora cordifolia stem aqueous extract of 2.06 g/100 mL. TCAE: Tinospora cordifolia stem aqueous extract; SLAE: Senna siamea leaf aqueous extract; SPAE: Senna siamea pods aqueous extract.

Table 2 Total phenolic content in Tinospora cordifolia stem aqueous extract, Senna siamea leaf aqueous extract and Senna siamea pods aqueous extract.

Samples	Round 1 (g/100 mL)	Round 2 (g/100 mL)
TCAE	2.06	2
SLAE	1.96	1.87
SPAE	1.39	1.1

In the following table, the total flavonoid concentration of Tinospora cordifolia stem aqueous extract, Senna siamea leaf aqueous extract and Senna siamea pods aqueous extract and control cells are estimated and higher total phenolic content is present in Tinospora cordifolia stem aqueous extract with 2.06 g/100 mL. TCAE: Tinospora cordifolia stem aqueous extract; SLAE: Senna siamea leaf aqueous extract; SPAE: Senna siamea pods aqueous extract.

Total tannin content: Total tannins were highest in SLAE (4.01 g/100 mL and 3.93 g/100 mL), followed by SPAE (2.40 g/100 mL and 2.39 g/100 mL) and TCAE (0.95 g/100 mL and 0.89 g/100 mL). (Figure 1C, Figure 4, Table 3).

Figure 4 Estimation of total tannin content. The above graph represents the concentration of total tannin content in the Tinospora cordifolia stem aqueous extract, Senna siamea leaf aqueous extract and Senna siamea pods aqueous extract. The highest tannin is found in Senna siamea leaf aqueous extract of 4.01 g/100 mL. TCAE: Tinospora cordifolia stem aqueous extract; SLAE: Senna siamea leaf aqueous extract; SPAE: Senna siamea pods aqueous extract.

Table 3 Total tannin content in Tinospora cordifolia stem aqueous extract, Senna siamea leaf aqueous extract and Senna siamea pods aqueous extract.

Samples	Round 1 (g/100 mL)	Round 2 (g/100 mL)
TCAE	0.95	0.89
SLAE	4.01	3.93
SPAE	2.40	2.39

In the following table, the total flavonoid concentration of Tinospora cordifolia stem aqueous extract, Senna siamea leaf aqueous extract and Senna siamea pods aqueous extract and control cells are estimated and higher total tannins content is present in Senna siamea leaf aqueous extract with 4.01 g/100 mL. TCAE: Tinospora cordifolia stem aqueous extract; SLAE: Senna siamea leaf aqueous extract; SPAE: Senna siamea pods aqueous extract.

Enzymatic activity

The total protein obtained for T. cordifolia stem was 1.7 mg, S. siamea leaf was 1.4 mg and S. siamea pods were 0.5 mg. The enzymatic activity of catalase, SOD, peroxidase, PPO and ascorbate oxidase were measured in protein extracts of T. cordifolia stem, S. siamea leaf and pods. Catalase activity was highest in the stem of T. cordifolia (161.42 μmol/minute), followed by S. siamea pods (30.41 μmol/minute) and S. siamea leaf (11.69 μmol/minute). The specific catalase enzyme activity was 94.95 μmol/minute/mg in T. cordifolia stem, 21.72 μmol/minute/mg in S. siamea leaf, and 23.38 μmol/minute/mg in S. siamea pods (Table 4).

Table 4 Specific activity of catalase enzyme in Tinospora cordifolia stem, Senna siamea leaf and Senna siamea pods.

Samples	Protein concentration (mg)	Enzyme activity of catalase (μmol/minute)	Specific activity (µmol/minute/mg)
Tinospora cordifolia stem	1.70	161.42	94.95
Senna siamea leaf	1.40	30.41	21.72
Senna siamea pods	0.50	11.69	23.38

In the following table, the specific activity and enzymatic activity of catalase enzyme in Tinospora cordifolia stem, Senna siamea leaf and Senna siamea pods are estimated and are found to be high in Tinospora cordifolia stem.

The SOD activity was highest in S. siamea pods (84.65 μmol/minute), followed by T. cordifolia stem (51.39 μmol/minute) and S. siamea leaf (15.11 μmol/minute). Specific SOD activity was 30.22 μmol/minute/mg in T. cordifolia stem, 60.46 μmol/minute/mg in S. siamea leaf, and 30.23 μmol/minute/mg in S. siamea pods (Table 5).

Table 5 Specific activity of superoxide dismutase enzyme in Tinospora cordifolia stem, Senna siamea leaf and Senna siamea pods.

Samples	Protein concentration (mg)	Enzyme activity of superoxide dismutase (μmol/minute)	Specific activity (µmol/minute/mg)
Tinospora cordifolia stem	1.70	51.39	30.22
Senna siamea leaf	1.40	84.65	60.46
Senna siamea pods	0.50	15.11	30.23

In the following table, the specific activity and enzymatic activity of superoxide dismutase enzyme in Tinospora cordifolia stem, Senna siamea leaf and Senna siamea pods are estimated and are found to be high in Senna siamea leaf.

Peroxidase activity was highest in T. cordifolia stem (1275 μmol/minute), followed by S. siamea pods (119.7 μmol/minute), and S. siamea leaf (17.76 μmol/minute). The specific peroxidase activity in T. cordifolia stem was 750 μmol/minute/mg, and 35.52 μmol/minute/mg in S. siamea pods (Table 6).

Table 6 Specific activity of peroxidase enzyme in Tinospora cordifolia stem, Senna siamea leaf and Senna siamea pods.

Samples	Protein concentration (mg)	Enzyme activity of peroxidase (μmol/minute)	Specific activity (µmol/minute/mg)
Tinospora cordifolia stem	1.70	1275	750
Senna siamea leaf	1.40	119.70	85.50
Senna siamea pods	0.50	17.76	35.52

In the following table, the specific activity and enzymatic activity of peroxidase enzyme in Tinospora cordifolia stem, Senna siamea leaf and Senna siamea pods are estimated and are found to be high in Tinospora cordifolia stem.

PPO activity was highest in S. siamea pods (122.35 μmol/minute), followed by T. cordifolia stem (91.07 μmol/minute) and S. siamea leaf (88.81 μmol/minute). The specific PPO activity was 53.57 μmol/minute/mg in T. cordifolia stem, 63.43 μmol/minute/mg in S. siamea leaf, and 244.70 μmol/minute/mg in S. siamea pods (Table 7).

Table 7 Specific activity of polyphenol oxidase enzyme in Tinospora cordifolia stem, Senna siamea leaf and Senna siamea pods.

Samples	Protein concentration (mg)	Enzyme activity of polyphenol oxidase (μmol/minute)	Specific activity (µmol/minute/mg)
Tinospora cordifolia stem	1.70	91.07	53.57
Senna siamea leaf	1.40	88.81	63.43
Senna siamea pods	0.50	122.35	244.70

In the following table, the specific activity and enzymatic activity of polyphenol oxidase enzyme in Tinospora cordifolia stem, Senna siamea leaf and Senna siamea pods are estimated and are found to be high in Tinospora cordifolia stem.

The ascorbate oxidase enzymatic activity was highest in T. cordifolia stem (5574.82 μmol/minute), followed by S. siamea pods (3256.13 μmol/minute) and S. siamea leaf (636.20 μmol/minute). The specific activity of ascorbate oxidase was 3279.30 μmol/minute/mg in T. cordifolia stem, 2325.80 μmol/minute/mg in S. siamea leaf, and 1272.40 μmol/minute/mg in S. siamea pods (Table 8).

Table 8 Specific activity of ascorbate oxidase enzyme in Tinospora cordifolia stem, Senna siamea leaf and Senna siamea pods.

Samples	Protein concentration (mg)	Enzyme activity of ascorbate oxidase (μmol/minute)	Specific activity (µmol/minute/mg)
Tinospora cordifolia stem	1.70	5574.82	3279.30
Senna siamea leaf	1.40	3256.13	2325.80
Senna siamea pods	0.50	636.20	1272.40

In the following table, the specific activity and enzymatic activity of ascorbate oxidase enzyme in Tinospora cordifolia stem, Senna siamea leaf and Senna siamea pods are estimated and are found to be high in Tinospora cordifolia stem.

Cell line study

The highest amount of cell death was induced by 200 μg of each extract (Figure 5). ROS activity significantly increased in cells treated with 150 μg of any of the three extracts (Figure 6). MMP decreased with increasing concentration of extract, but highly decreased upon 150 μg treatment of any of the three extracts (Figure 7). TCAE, SLAE or SPAE treatment induced THP-1 cell cycle arrest. TCAE had the strongest effect, with 56% and 56.4% of cells in G0/G1, while SLAE induced G0/G1 in 47.1% and 47.4% and SPAE induced 51% and 52.5% of cells in G0/G1. There was a concomitant decrease in the proportion of cells in stage G2/M, with 12.4% and 11.9% after TCAE treatment, 22.4% and 21.5% after SLAE treatment, and 15.3% and 14.6% after SPAE treatment (Figure 8, Table 9). One-way ANOVA analysis of the cell cycle assay showed a significant difference between treatments, F (2,3) = 96.12, P = 0.0019, ƞ² = 0.98. Treatment with TCAE, SLAE or SPAE induced early apoptosis (Figure 9, Figure 10).

Figure 5 Cell viability in THP-1 cells treated with Tinospora cordifolia stem aqueous extract, Senna siamea leaf aqueous extract and Senna siamea pods aqueous extract. A: The following graph shows cell viability after treatment with 50-200 μg Tinospora cordifolia stem aqueous extract (TCAE). The highest cell cytotoxicity is found in 200 μg TCAE treatment; B: The following graph shows cell viability after treatment with 50-200 μg Senna siamea leaf aqueous extract (SLAE). The highest cell cytotoxicity was found in the 200 μg SLAE treatment; C: Cell viability in THP-1 cells treated with Senna siamea pods aqueous extract (SPAE). The following graph shows the cell viability after treatment with SPAE of concentration ranging 50-200 μg. The highest cell cytotoxicity is found in TCAE treatment with 200 μg concentration. ^aP < 0.05 vs control group; ^bP < 0.01 vs control group; NS indicates not significant. TCAE: Tinospora cordifolia stem aqueous extract; SLAE: Senna siamea leaf aqueous extract; SPAE: Senna siamea pods aqueous extract.

Figure 6 Reactive oxygen species assay in THP-1 cells treated with Tinospora cordifolia stem aqueous extract, Senna siamea leaf aqueous extract and Senna siamea pods aqueous extract. A: The following graph shows the reactive oxygen species (ROS) activity in the THP-1 cells after treatment with Tinospora cordifolia stem aqueous extract (TCAE). Higher ROS activity is found in the TCAE 150 μg concentration treatment in the cells; B: The following graph shows the ROS activity in the THP-1 cells after treatment with Senna siamea leaf aqueous extract (SLAE). Higher ROS activity is found in the SLAE 150 μg concentration treatment in the cells; C: The following graph shows the ROS activity in the THP-1 cells after treatment with Senna siamea pods aqueous extract (SPAE). Higher ROS activity is found in the SPAE 150 μg concentration treatment in the cells. ^aP < 0.05 vs control group; ^bP < 0.01 vs control group; NS indicates not significant. TCAE: Tinospora cordifolia stem aqueous extract; SLAE: Senna siamea leaf aqueous extract; SPAE: Senna siamea pods aqueous extract.

Figure 7 Mitochondrial membrane potential assay in THP-1 cells treated with Tinospora cordifolia stem aqueous extract, Senna siamea leaf aqueous extract and Senna siamea pods aqueous extract. A: The following graph shows the mitochondrial membrane potential (MMP) activity in the THP-1 cells after treatment with Tinospora cordifolia stem aqueous extract (TCAE). The MMP activity is found to decrease with increase in the TCAE concentration. MMP is found to be decreased highly in 150 μg TCAE concentration treatment in the cells; B: The following graph shows the MMP activity in the THP-1 cells after treatment with Senna siamea leaf aqueous extract (SLAE). The MMP activity is found to decrease with increase in the SLAE concentration. MMP is found to be decreased highly in 150 μg SLAE concentration treatment in the cells; C: The following graph shows the MMP activity in the THP-1 cells after treatment with Senna siamea pods aqueous extract (SPAE). The MPP activity is found to decrease with increase in the SPAE concentration. MMP is found to be decreased highly in 150 μg SPAE concentration treatment in the cells. ^aP < 0.05 vs control group; NS indicates not significant. TCAE: Tinospora cordifolia stem aqueous extract; SLAE: Senna siamea leaf aqueous extract; SPAE: Senna siamea pods aqueous extract.

Figure 8 Cell cycle assay in THP-1 in the cells treated with Tinospora cordifolia stem aqueous extract, Senna siamea leaf aqueous extract and Senna siamea pods aqueous extract. The following figure shows the treatment of THP-1 cells with Tinospora cordifolia stem aqueous extract (TCAE), Senna siamea leaf aqueous extract (SLAE) and Senna siamea pods aqueous extract (SPAE) individually in the cells. The cells are arrested in the G0/G1 stage. A high number of cells are arrested upon treatment with TCAE, followed by SPAE and SLAE in flow cytometric analysis.

Figure 9 Apoptosis assay in cells treated with Tinospora cordifolia stem aqueous extract, Senna siamea leaf aqueous extract and Senna siamea pods aqueous extract. The following figure shows the induction of apoptosis with treatment of Tinospora cordifolia stem aqueous extract, Senna siamea leaf aqueous extract and Senna siamea pods aqueous in the THP-1 cells. Treatment with the extracts resulted in entry of the cells into early apoptosis in flow cytometric analysis.

Figure 10  Cells treated with Tinospora cordifolia stem aqueous extract, Senna siamea leaf aqueous extract and Senna siamea pods aqueous extract and untreated cells. A: The untreated THP-1 cells; B: The THP-1 cells treated with Tinospora cordifolia stem aqueous extract; C: The THP-1 cells treated with Senna siamea leaf aqueous extract; D: The THP-1 cells treated with Senna siamea pods aqueous extract. TCAE: Tinospora cordifolia stem aqueous extract; SLAE: Senna siamea leaf aqueous extract; SPAE: Senna siamea pods aqueous extract.

Table 9 Cell cycle analysis with the treatment with Tinospora cordifolia stem aqueous extract, Senna siamea leaf aqueous extract and Senna siamea leaf aqueous extract.

Sample name	% of the cells in different phases of cell cycle
	G1	G0/G1	S	G2/M
Control	1.36	45.20	31.70	21.60
	1.48	46.90	31.20	20.20
TCAE	2.12	56.00	29.10	12.40
	2.35	56.40	28.90	11.90
SLAE	1.54	47.10	28.80	22.40
	1.66	47.40	29.30	21.50
SPAE	1.58	51.00	31.60	15.30
	2.11	52.50	30.40	14.60

In the following, the data of the cell cycle analysis upon treatment with Tinospora cordifolia stem aqueous extract, Senna siamea leaf aqueous extract and Senna siamea pods aqueous extract and control cells are presented with the percentage of cells in the G1, G0/G1, S and G2/GM phase are presented in the two rounds of treatment in comparison to the control. TCAE: Tinospora cordifolia stem aqueous extract; SLAE: Senna siamea leaf aqueous extract; SPAE: Senna siamea pods aqueous extract.

Statistical analysis

One-way ANOVA analysis of total flavonoid compound content estimation was statistically significant [F (2,3) = 15.84, P = 0.025, ƞ² = 0.91]. One-way ANOVA analysis of total phenolic compound estimation was statistically significant [F (2,3) = 22.51, P = 0.015, ƞ² = 0.93]. One-way ANOVA analysis of total tannin compound content estimation was significant [F (2,3) = 2764.109, P < 0.001, ƞ² = 0.99].

DISCUSSION

We analyzed the phytochemicals and enzymatic activity of T. cordifolia stem and S. siamea leaf and pods, followed by in vitro analysis to identify anti-leukemic properties of the extracts. Total flavonoid content was high in SLAE, followed by SPAE and TCAE. Total phenolic compound content was highest in TCAE, followed by SLAE and SPAE. Total tannin content was high in SLAE, followed by SPAE and TCAE. One-way ANOVA analysis of the phytochemical compounds, flavonoids, phenolic compounds, and tannins in TCAE, SLAE and SPAE revealed a statistically significant difference (P < 0.05). We also studied the specific enzymatic activity of catalase, SOD, peroxidase, PPO and ascorbate oxidase. The T. cordifolia stem had the highest catalase activity and specific activity than S. siamea leaf and pods. For SOD enzymatic activity and specific activity, S. siamea leaf was more active than the T. cordifolia stem. T. cordifolia stem had higher peroxidase activity, followed by S. siamea pods and S. siamea leaf. The PPO activity and specific activity was high in the protein extract of S. siamea pods compared with T. cordifolia stem and S. siamea leaf. The specific activity and enzymatic activity of ascorbate oxidase was higher in the T. cordifolia stem than in S. siamea leaf or pods. The presence of the phytochemical compounds like flavonoid, phenolic compounds and tannins in TCAE, SLAE and SPAE gave them potential in treating ML, as these compounds have many therapeutic activities like antioxidative effects, induction of apoptosis, anti-proliferative, cytotoxic and anti-angiogenic effects. Enzymes like catalase, SOD, peroxidase, PPO and ascorbate oxidase also have significant anticancer roles, as these enzymes can detoxify hydrogen peroxide, modulate ROS levels, break down harmful compounds, induce oxidative stress and death of tumor cells, inhibit proliferation and migration of tumor cells and promote apoptosis. The presence of these enzymes in T. cordifolia stem, and S. siamea leaf and pods made them potentially useful for treating ML. In cytotoxicity assays, 200 μg of extract had the highest toxicity. ROS activity increased with increasing extract concentration and was higher in 150 μg. MMP assays decreased as extract concentration decreased, and the largest decrease was achieved with 150 μg extract, with cells arresting in the cell cycle and entering the early apoptosis. Increased ROS activity explains the induction of oxidative stress, which could result in a decrease in MMP due to damage of mitochondrial proteins and other components that lead to damage of the electron transport chain, causing a reduction of the proton gradient and depolarization of the mitochondrial membrane. The loss of MMP causes a release of proapoptotic factors, causing apoptotic pathway activation.

In this study, TCAE, SLAE and SPAE had potential therapeutic activities against ML in an in vitro study of THP-1 cells. However, further animal and clinical studies are required. The present investigation aimed to determine whether performed TCAE, SLAE or SPAE had any therapeutic effect in leukemia. Further studies using positive controls like cytarabine, idarubicin chloride and others in comparison to T. cordifolia stem and S. siamea leaf and pods are required in both in vitro and animal models. The present investigation also explored whether any of the three extracts had apoptotic effects on cells. Indeed, treatment with the extracts caused an increase in ROS activity, which lead to decreased MMP and cell death. However, further study is required to estimate the proteins related to apoptosis, like Bcl-2 and caspase proteins, after treatment in animal models.

T. cordifolia has been studied against different cancers. Methanolic extract of the stem had a significant anti-cancer effect of cytotoxicity against the breast cancer cell line MDA-MB-231[24]. The extract of T. cordifolia inhibited population assay in a dose-dependent manner, and the dichloromethane and petroleum ether T. cordifolia extract inhibited transporters ABC-G2 and ABC-B1 in malignant tumors and made tumor cells sensitive to cytotoxicity with chemotherapeutic drugs[25]. Berberine compound in T. cordifolia has an anti-proliferative effect. Administration of HCA-7 human colon adenocarcinoma cells with this compound downregulated the expression of 33 genes, which were involved in cell cycle, differentiation and epithelial-mesenchymal transition in cell line by reverse transcription polymerase chain reaction analysis[26]. In the oral cancer cell line AW13516, treatment with the T. cordifolia aqueous extract induced apoptosis by caspase activity and decreased the expression of EMT genes in a dose-dependent manner[27]. Ethanolic extract of T. cordifolia induced inhibition of cell proliferation of C6 glioma cells and induced cell differentiation, showed anti-migratory and anti-invasive potentials and arrested the cell cycle in G0/G1 and G2/M phase[28]. In dimethyl hydrazine-induced colorectal cancer in Sprague Dawley rats, treatment with T. cordifolia extract downregulated the expression of NF-κB, MMP-9, TNF-α, IL-6 and COX-2 but enhanced the pro-apoptotic caspase, IL-12 and antioxidant activity[29]. In MCF-7 cells, T. cordifolia treatment showed anti-proliferation activity, and cell viability decreased in a dose-dependent manner[30]. A murine model induced with Dalton’s Lymphoma was treated with T. cordifolia extract, which resulted in reduced neutrophil count in the peripheral blood and reduced neutrophil infiltration in vital organs and induced neutrophil hyperactivation by decreasing the expression of genes like MPO, NE, MMP-8, and MMP-9 and CSTG in the early and mid-stage of tumor growth[31].

S. siamea is a common medicinal plant. A previous study examined its therapeutic properties on R. raninus and S. cerevisiae. Treatment caused severe DNA damage and subsequent cell death[9]. Barakol is a major compound in S. siamea, and it has anticancer and antioxidant properties. Barakol inhibits MMP-3 activity and thus exhibited anti-metastatic and inhibited cell migration, but was found to be non-toxic to normal cell lines[32]. Emodin and sennamin B, two compounds isolated from S. siamea, were found to have anti-tumor effects on mouse skin tumors, which were induced by 7,12-dimethylbenz(a) anthracene and 12-O-tetradecanoylphorbol-13-acetate[33].

ROS is extensively studied in human diseases, particularly as cancer, and is a byproduct of cellular processes that use oxygen for metabolism. Increased ROS levels damaged proteins, nucleic acids, lipid membranes and organelles, which leads to activation of apoptosis in cells. ROS was produced during chemotherapy treatment for cancer, which could alter redox homeostasis in cells[34]. A triterpenoid compound, pristimerin, increased intracellular ROS levels that controlled ER stress and the AKT/GSK3β signaling pathway in TE-1 and TE-10 esophageal cancer cells[35]. The compound biochanin A and sulforaphane showed apoptotic activity in MCF-7 cells by increasing ROS production[36].

In cancer, MMP is abnormally high, which is associated with the invasive properties of cancer cells and increased metastasis. It was mainly regulated by intracellular mechanisms, such as mitochondrial membrane, both outer and inner membrane ion transporters, cytoskeletal elements, and biochemical signaling pathways[37]. Artesunate is an antimalarial drug, and treatment of SiHa cells for esophageal and gastric cancers decreased MMP, as indicated by a decrease in cell proliferation and Bcl2, Bcl-xl and Mcl-xl levels[38]. In HeLa and CaSki cervical cancer cells, different concentrations of curcumin showed a decrease in MMP, which was evaluated by staining with JC-1[39]. Caffeic acid is an effective compound in traditional Chinese herbal medicine, and in triple-negative breast cancer, it inhibited proliferation and disrupted MMP[40]. In breast cancer, polyphyllin VII induced apoptosis, inhibited proliferation, increased ROS and decreased MMP. This compound also downregulated SOS1 and inhibited the MAPK/ERK pathway in an in vivo study in nude mice[41].

Cell cycle arrest is one approach to treating cancer. It provides the time to repair damaged DNA and was a characteristic of therapies like radiation and chemotherapy. N-Hexane extract of Commiphora myrrha induced an inhibition in breast cancer MDA-MB-231 and MCF-7 cells at the G0/G1 phase and also induced apoptosis and downregulated the pathway of Cyclin D1/CDK4-Rb[42]. Functional tea extracts treatment in human hepatocellular carcinoma cells induced apoptosis, and the cells were arrested at the G0/G1 phase, with decreased the telomerase activity[43].

The present investigation had several limitations. It mainly focused on identifying the therapeutic effect of T. cordifolia stem, S. siamea leaf and pods using an in vitro model for ML. The limitation of the study was that it lacked an in vivo study to investigate the effect of T. cordifolia stem and S. siamea leaf and pods extracts, which will be carried out further. The present study also lacked the use of a positive control in comparison to the treatment of the extracts TCAE, SLAE and SPAE in the in vitro model THP-1, which required further investigation. The study also lacked quantification of the apoptosis-related protein upon treatment with the three extracts, which also needed to be carried out further. The study also had limitations in the identification of the compounds present and also in the identification of the active compounds that have possible anticancer properties against ML.

The future direction of the present study is that TCAE, SLAE and SPAE extracts need to be further explored to identify the compounds that have possible therapeutic effects and to investigate animal models to study their therapeutic properties against ML. Further clinical studies should evaluate the potential of these three extracts in the treatment of ML.

CONCLUSION

The present study was performed to analyze the phytochemical compounds and enzymatic activity in the T. cordifolia stem and S. siamea leaf and pods and to carry out an in vitro analysis in THP-1 cells to study anti-leukemic effect. In the investigation, TCAE, SLAE and SPAE were found to have therapeutic properties due to the presence of phytochemical compounds like flavonoids, phenolic compounds and tannins and also the activity of the enzymes catalase, SOD, peroxidase, PPO and ascorbate oxidase, which contributed to therapeutic activity against ML. TCAE, SLEA and SPAE were used further to study anti-leukemic effect, in which all three extracts showed better performance with increased ROS activity, reduced MMP, resulting in apoptosis induction and G0/G1 arrest. From the above study, it could be indicated that TCAE, DLAE and SPAE have potential therapeutic activities against ML and could be a possible treatments for ML, but further clinical studies are required.

ACKNOWLEDGEMENTS

The authors are thankful to their respective Universities and Institutions for their valuable support.