The effect of coconut extract on callus growth and ultrasound waves on production of betulin and betulinic acid in in-vitro culture conditions of Betula pendula Roth species

To determine the effect of coconut extract on callogenesis of Betula pendula, Roth stem barks were cultured in NT (Nagata and Takebe) basic culture media in two individual experiments: i) cultivation explant in different treatments of coconut extracts combined with 1 mg l-1 2, 4-D (2, 4-Dichlorophenoxyacetic acid) and ii) callogenesis in NT media containing 1.5 mg l-1 2,4-D and 0.5 mg l-1 BAP (6-Benzylaminopurine) and then cultivation under the first experiment treatments. The first experiment demonstrated that not all concentrations of coconut extracts lead to callus induction individually, but callus induction increased 84% in a culture containing 5% coconut extract plus 1 mg l-1 2, 4-D. Based on the results of the second experiment, this treatment also significantly increased the wet and dry weights of the produced calluses. The possibility of increasing the betulinic acid and betulin by ultrasound was also studied. Samples cultivated in the selected culture medium were exposed to ultrasound waves in two forms of 1) one exposure and 2) twice exposure (repetition with 24 hr interval) in steps of 20, 60, 100, and 160 sec, and one treatment as the control. Betulin and the betulinic acid amount were determined using HPLC (High-Performance Liquid Chromatography). The maximum betulinic acid and betulin were obtained in 100 sec in once exposure (2.3 mg g-1 DW) and 160 sec in the twice exposure (0.75 mg g-1 DW) to ultrasound. The results showed that betulinic acid content was more than betulin

Increased Induction of Apoptosis in ESCC (Esophageal Squamous-Cell Carcinoma) by Betula pendula Roth Stem Cell Extract Containing Triterpenoids Compared to Doxorubicin

Background: Esophageal Squamous-Cell Carcinoma (ESCC) is one of the most life-threatening malignancies worldwide, with a growing incidence in Iran higher than the global average. Objective: The present study, for the first time under patent number (97668), introduces a method using in vitro production of activated-Birch stem cells using biotechnological techniques of tissue culture and plant stem cell culture from Betula pendula Roth (Birch) bark. Methods: In the first step, Birch stem cells were produced in large amounts using tissue culture, and then the amount of triterpenoids of its extract was measured by the HPLC method. In the second step, the cytotoxicity was evaluated by MTT, and the IC50 was calculated. The cellular apoptosis in response to the extract compared to doxorubicin was measured using the Annexin V kit and the flow cytometry method. Results: The optimized method introduced in the current study efficiently produced plant stem cells containing triterpenoids in large quantities over a period of 2-4 months. Our findings indicated that the growth of ESCC cells decreased by induction treatment 3 times (24, 36, 48 hours). IC50 values were obtained in 24 hours for the natural bark extract, Birch stem cell extract, doxorubicin and interactions of two extracts with doxorubicin at 300μg/mL, 1700μg/mL, 0.5μM, 150μg/mL, 1800μg/mL, respectively. In the flow cytometric test, the Birch stem cell extract showed the highest percentage of apoptosis, with 92.5% for total apoptosis. The percentage of total apoptosis in doxorubicin treatment was 85.33%, and the combination of doxorubicin with Birch stem cell extract was 88.33%. Natural bark extract and its combination with a lower percentage (69.33% and 70.33%, respectively) caused apoptosis of esophageal cancer cells. Conclusion: Owing to the extinction of Birch in Iran and its inaccessibility and exploitation, Birch stem cells can be cultured as an appropriate alternative source to produce valuable triterpenoids for pharmaceutical purposes. Additionally, according to the results of this study, stem cells can be used to enhance the treatment of esophageal cancer and supplementation with chemotherapy. </jats:sec

Improved accumulation of betulin and betulinic acid in cell suspension culture of <i>Betula pendula</i> roth by abiotic and biotic elicitors

Betulin (B) and betulinic acid (BA) are two triterpenes with diverse pharmacological and physiological actions. Elicitation of Betula pendula Roth cell cultures by elicitors is an excellent strategy to increase B and BA levels. Six abiotic and biotic elicitors were studied to improve accumulation of B and BA in the cell culture of B. pendula. The B and BA production in treated cells was verified by HPLC. The results showed the maximum growth index (7) on day 3 in cells treated with 0.5 mg L−1 chlorocholine chloride (CCC). The increased accumulation of BA in the cells treated with 200 mg L−1 of chitosan was found to be 5.9 × (6.5 mg g−1 DW) higher over control cells. Treating the cells with 2 mg L−1 of CCC, after 7 days, led to 149.3× enhancement of B content (19.4 mg g−1 DW) over the controls. Production of this triterpenoid at a much shorter time with a much higher growth rate can be economic and lead to producing large amounts of B and BA for anti-cancer and HIV drugs preparation.</p