EFFECT OF LYOPHILIZATION AND CRYOPRESERVATION ON PLANT LEAVES OF TERMINALIA ARJUNA TERMINALIA CATAPPA, TERMINALIA CHEBULA, JATROPHA GOSSYPIFOLIA, JATROPHA CURCAS

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·         Chemical constituents and components
Terminalia chebula contains the triterpenes arjun glucoside 1, arjungenin and the chebulosides 1&2. Other constituents contains tannins up to 30%, chebulic acid 3-5%, chebulinic acid 30%, tannic acid 20-40%, ellagic acid, 2,4-chebulyi–β-D-gluco pyranose, gallic acid, ethyl gallate, punicalagin terflavin A , terchebin, some purgative of the nature of anthraquinone , flavonoids like luteolin, rutins, and quercetin etc [SuryaPrakash D et al., 2012].

·         Geographical location
T. chebula occurs scattered in teak forest, mixed deciduous forest, extending into forests of comparatively dry types. It grows in India, Myanmar, Bangladesh, Iran, Egypt, Turkey, China etc. In India Haritakitree is grows in deciduous forests and found in North India and South words to the Deccan table lands at 1000 to 3000 ft. In Myanmar country grows up to 5000 ft Terminalia chebula plant is a native plant in India and Southeast Asia and is extensively cultivated in Taiwan. [SuryaPrakash D et al., 2012;Said M et al., 2012].

·         Medicinal uses
It is used to increase the appetite, as digestive aid liver stimulant, as stomachic, as gastro-intestinal prokinetic agent and as mild laxative. It is indicated in protracted diarrhea with hematochezia and prolapse of rectum. It is a good nervine, used in nervous weakness, nervous irritability.It is helpful in renal calculi, dysurea, and retention of urine and used for treating parasitic infection. It is used as a blood purifier, gargle for sore throat, ulcerated gums, and muscular rheumatism [SuryaPrakash D et al., 2012].

D. Jatropha gossypifolia
It is belongs to the family Euphorbiaceae and the order, “Geraniale”.The common name for J. gossypifolia is bellyache bush, pignut or fignut, and in Yoruba land it is commonly known as “Lapalapa”. It is a bushy, gregarious shrub, up to 1.8m, 3-5 lobed, approximately 20 cm long and wide, with leaves having a long petiole, covered with glandular hairs. The seed are greenish capsule-like seeds. The leaf stalks are covered with coarse dark brown hairs and the young leaves are sticky. It has thin, often greenish bark, which exudes copious amount of watery sap when cut. The fruits are three-celled with one seed per cell. J. gossypifolia is widely cultivated as ornamental plant. It is the common red species planted around houses. It is also planted to control the soil erosion along the slopes. It prefers arid environment [Seth R and Sarin R, 2010].

 

Figure 4: Jatropha gossypifolia

·         Binomial name Jatropha gossypiifolia

·         Scientific classification
Kingdom
: Plantae
Order:       Geraniale
Family:     Euphorbiaceae
Genus:      Jatropha
Species:     J. gossypiifolia

·         Chemical constituents and components
The major phytochemicals of interest are alkaloids, tannins, flavonoids, phenolic compounds, and steroidal saponins; however, other diverse groups of naturally occurring phytochemicals such as unsaturated sterols, triterpenoids, and essential oils are also present [Seth R and Sarin R, 2010].

•    Geographical location
Native to tropical America, but is now cultivated widely in tropical countries throughout the world. It is grown occasionally in warmer parts of Australia and is naturalised in a few places in   Queensland and the Northern Territory.  In Florida it is found chiefly south of Orlando.  It is also a common plant in the   Hawaiian Islands.  Introduced to southern Africa, the plant has spread from Mozambique through Zambia to the Transvaal and Natal. This species is also found throughout the warmer parts of Asia. This plant is normalized in many parts of India. It has also naturalized in most tropical area of the world [Jadhav A et al., 2012].

·         Medicinal uses
The leaves of J. gossypifolia are used for intermittent fevers, carbuncles, eczema, itches, sores on the tongues of babies, swollen mammae, stomachache, and venereal disease. The leaf decoction is used for bathing wounds [Oduola T et al., 2007].

E. Jatropha curcas
Jatropha curcas L belongs to the euphorbia family. It is a large coarse annual shrub or small short lived tree which can grow 3.5 to 4.5 metres (8-15 feet) tall.  It has thin, often greenish bark which exudes copious amounts of watery sap when cut. Leaves are dark green; alternate, simple, ovate to slightly lobed with 3-5 indentations. Petioles are 10cm (4 inches) long. Flowers are yellow to green in color, borne in axils of the leaves and being small is mostly hidden by foliage. Fruit are small capsule-like, round fruit; about 2.5 - 4 cm (1-1.5 inches) in diameter.  These are green and fleshy when immature, becoming dark  brown when ripe  and splitting to release 2 or 3 black seeds each about 2 cm (3/4 inch) long.  The meat of the seeds is white and oily in texture and is reported to have an agreeable taste. [inchem.org/documents/pims/plant/jcurc.htm]


Figure 5: Jatropha curcas

·         Binomial name: Jatropha curcas
·         Scientific classification
Kingdom
: Plantae
Order
:       Malpighiales
Family:    
Euphorbiaceae
Genus:
      Jatropha
Species:
     J. curcas

·         Chemical constituents and components
The phytochemicals analysis of the extract revealed the presence of triterpenoids, volatile oils, alkaloids, flavonoids, saponins and tannins [Uche F and Aprioku J, 2008].

•    Geographical location
It is widely cultivated as an ornamental. Native to tropical America, but is now cultivated widely in tropical countries throughout the world. It is grown occasionally in warmer parts of Australia and is naturalised in a few places in   Queensland and the Northern Territory.  It has been planted in Gujarat in Ahmedabad, Kutch, Bhavnagar, Jamnagar, Surat, Banaskantha, Rajkot, and Surendranagar. It is one of the 18 species found scattered in various parts of India [Punia M, 2007].

·         Medicinal uses
Most parts of this plant are used for the treatment of various human and veterinary ailments. The white latex serves as a disinfectant in mouth infections in children.The leaves contain apigenin, vitexin and isovitexin etc. which along with other factors enable them to be used against malaria, rheumatic and muscular pains [Thomas R et al., 2008].

METHODOLOGY

COLLECTION OF SAMPLE
Total 5 plant species were collected for herbarium preparation as well as DNA isolation from Indroda Botanical Garden, Gandhinagar [Figure 4.3.1]. For herbarium preparation the plant specimen was placed on a filter paper. The leaves were arranged to avoid overlapping and the respective filter papers were kept in the herbarium press.

Table 1: List of plant species

Plants name

Family

Latitude

Longitude

Terminalia arjuna

Combretaceae

N 23° 11.650

E 72° 38.853

Terminalia catappa

Combretaceae

N 23° 11.659

E 72° 38.861

Terminalia chebula

Combretaceae

N 23° 11.722

E 72° 38.936

Jatropha curcas

 Euphorbiaceae

N 23° 11.665

E 72° 38.809

Jatropha gossypifolia

 Euphorbiaceae

N 23° 11.664

E 72° 38.809

HERBARIUM PREPARATION
1.      The plant sample which was earlier collected from the field was taken out from the herbarium press.
2.      The plant samples were treated with mercuric chloride solution.
3.      The treated plant samples were placed in a new blotting paper and name of the plant, date of collection and name of the collector were written.
4.      All the other plant samples were treated similarly and put in the herbarium press.
5.      The blotting paper was changed every alternate day till the plant samples were dried completely.
6.      After the samples were dried, they were taken out from the herbarium press and stuck on the herbarium sheet with the help of fevicol.
7.       The details about the plant were filled on the herbarium sheet.

CRYOPRESERVATION

Surface sterilization
Leaves were sterilized by surface washing under tap water than distilled water to remove dust and other contaminants.

Weighing of sample
The leaves were dried with blotting paper and approximately 20g leaves were weighed without middle ribs.

Sample preparation for cryopreservation
Leaves were crushed into liquid nitrogen to make a fine powder and 3 cryovials were filled up for each plant and were cryopreserved at -196°C cryopreservation was carried out by placing the cryovials in canisters and immersing them in the cryocan containing liquid nitrogen. DNA extraction was carried out from cryopreserved sample at intervals of 1, 7 and 15 days.

LYOPHILIZATION

Surface sterilization
Leaves were sterilized by surface washing under tap water than distilled water to remove dust and other contaminants.

Sample preparation for lyophilization
The leaves were dried with blotting paper and middle ribs were removed. They were crushed into liquid nitrogen to make fine powder and approximately 15g were weighed. After lyophilization, the powder was collected in petridish which were sealed with cling film.

Lyophilization process
Plant samples were lyophilized at vacuum of 0.16 mbar and -500C for 72 hrs. Lyophilized powder was stored in tightly sealed petridish at room temperature.

The Super Modulo freeze dryer used for lyophilization at GSBTM does not facilitate the temperature and pressure regulation. The protocol for operation of this instrument was as follows:
1. The powdered samples were frozen for some time at -20°C freezer.
2.  The frozen leaves were evenly spread on the product trays without the delay for the leaves to cool down again.
3. The racks with the samples were placed in the dome and covered with the acrylic lid.
4. It was made sure that all the detachable parts associated with vacuum are well greased so as to minimize the chances of any leakage.
5. The “Fridge” switch was turned on. Within 1 hr, the chamber temperature should be around -40°C to -50°C.
6. The “pump” switch was turned on. The pressure reached up to 10-1 mbar.
7. The samples were lyophilized for 1 whole day.
8. After the run was complete, the “Pump” switch was turned off. The knobs were gradually opened to release the pressure.
9. The samples were placed in a dry, air tight environment as soon as possible and stored out of direct sunlight [Super Modulo Instruction Manual, 2006].

Residual moisture content:
The moisture content was determined using following formula.

Moisture content (%) = (fresh weight – dry weight) ×100
                                        fresh weight

Residual moisture content (%) = 100- Moisture content

DNA EXTRACTION
DNA extraction was carried out from cryopreserved, lyophilized and oven dried plant samples using two different protocols which are mentioned below as follows:

A. CTAB protocol [Doyle J and Doyle J, 1987]

  1. Water bath was set to 65 ºC and CTAB buffer was put into the water bath.
  2. 100 mg of plant material (cryopreserved, lyophilized, oven dried samples) was taken in a motor pestle.
  3. Liquid nitrogen was added and the sample was crushed to powder form. (In case of already crushed samples, proceed to next step)
  4. 3 ml of 3% CTAB buffer, containing 6% PVP (pH 8) and 6.1 µl of β-mercaptoethanol was added.
  5. The solution was incubated at 65ºC for 1 hour.
  6. The tube was centrifuged at 12,000 rpm for 5’ at 4 0C by using centrifuge (Eppendorf).
  7. The supernatant was collected in fresh tube and 5µl/ml of Proteinase K was added and incubated at 65 °C for 1 hr.
  8. Equal volume of P: C: I was added, mixed gently, centrifuged 12000 rpm for 15 mins.
  9. Supernatant was collected, equal volume of C: I was added, mixed and centrifuged at 12000 rpm for 15 mins.
  10. Step 9 was repeated.
  11. 5 µl of 20 mg/ml RNase A or 10µl of 10 mg/ml RNase A was added, incubated at 65 °C for 1 hour or 37 °C overnight.
  12. 1/10th volume of 3M sodium acetate and equal volume of absolute ethanol was added, incubated for 20 mins at -20°C.
  13. The samples were centrifuged at 12000 rpm for 10 mins, supernatant was discarded and the pellet was recovered.
  14. The pellet was washed twice with 70% ethanol and centrifuged at 10000 rpm for 10 mins.
  15. The pellet was allowed to air dry and dissolved in 200 µl of 1X TE buffer.

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