Journal of Scientific & Indu strial Research

Vol. 62, May 2003 , pp 457-461

Pilot Plant Processing Data for the Isolation of Arteniisinin from the New Variety of Artemisia annua 'Jeevanraksha'

S Tandon*, A P Kahol, D C Jain, R S Bhakuni and Sushil Kumar

Central Institute of Medi cinal and Aromati c Plants, P .O. CIMAP Campus , Lu cknow 226 0 I 5

Received: 03 June 2002; revi sed received : 17 February 2003, accepted: 28 February 2.003

Arteillisia al7l7l1a, known, as 'qinghao' in China is a major source of artemi sinin, used for the synthesis of many potent drugs effec ti ve against malari a caused by chloroqunine sen sitive and resistant strain o f Plaslllodiftln/cilcipamJlI. CIMAP has recently developed a new vari ety of A r telllisia alllZl.ta ' Jeevanraksha ' containing hi gh levels o f artemi sinin. Processing parameters for an impro ved and economi c process for the isolation of artemisinin from Artelllisia a l/I/II({ have been stand ardi sed that are useful for commercial equipment design.

Introduction

Malaria still remains one of the world ' s most devastating human infections affecting over 500 milli on people and causing more than two million death s each yea r. In Indi a al so malari a is a major health problem. The currentl y ava il able drug in the market based on quinine has become ineffective due to res istance deve loped by the several species of Plasl11 odium . Cerebral malaria is the most seri ous manifestation of P. fa lcip(frum infec ti on related with acute meningit is, acute delerium, comatose state and finall y leadin g to death of host. A rtem isia allllua (Q inghao) is a Chinese herb ll sed for treating malaria. At present arteilli sin in based de ri vati ves are the onl y effect ive drugs aga inst cerebral and mul ti -drug res istant ma lari a. The total sy nthes is of th is molecu le e1 0es not of fer an efficient way of production and therefore the pl ant remains the onl y source of arte lll is inin . Va rious de rivati ves have been prepared from artemi sin ill such as artemelher, arteether and artesu nate. They are effecti ve whell gi ve n orally , suppos itory and intramusculal". '

!\ r/t'Jll isiu iJlllIlIU plant \vas illtrociuceci in Ind ia by CHvlAP in 1981') ,mel <llLlp tcd for cultivatio ll in ](,lshill ir v,t1!c\. For illdl!.~tri:tI productio ll of <lrtell1isi ni ll . it "·dS neccss~lry to get e!1ough we ll

growing plants with high artemi sinin content. A new variety of Artemisia allilua ' l eevanraksha' having a higher content of artemi sinin in the range of 0.6 -1 .2 per cent has been recentl y released by CIMAP2. CIMAP has al so developed an effici ent improved process for the isolation of artemisinin from A rtemisia [lJIllUa and a process fo r simultaneous extraction of essential oil and isolat ion of artemi sill in from the plant A rtemisia annua' A. CIMAP has also developed the process technology for conversion of artemisinin to arteether. 5 Data on the production of artemisinin from the plant Artemisia OIllZLIO on pil ot scale is scarce, therefore an experimental programme for productio ll of artemi sini n on pil ot-scal e was taken up and is reported in th is paper.

Mater ials and Methods

Plant Material

The experimental plants of Artelll isia ( 1I1Jl IJU c\'. leevanraksha were grown under SUbt ropical agro­cli mate at CII\1AP farm, Lucknow in 1998-99 and 1999-2000 win te r-sul1lmer-\l1ol1soon seasons. Each year, a nursery or the plan ts was developed by sowillg the seeel s ill the midelle of Deccmbcl". Seed l ill~s were transplanted to maill fi eld in :') X -+ 1112 bed; ill the second week of February at a density of about 50,000 plants/ha and the plots had been app lied wit h N, P and K at 80, 40 and 40 kg/ha, re:'pecl ive ly. Tht: crop was irr igated as and when required.

458 J SC I INO RES VOL 62 MAY 2003

Harvesting otArtemisia annua

C rops of A. alZlZua transplanted in the second week of February in 1999 and 2000 were ratooned and ha rves ted severa! times. W he reas the crop of 1999 was harv~sted three ti mes- third weeks of May, July and second 'week of October; the c rop of 2000 was harvested fOLlr t i mes-thi I'd wee k of May , second weeks o f June, August and September. T he harvested materi a l was shade dr ied and leaves separated and we ighed .

Est i ma tion of A rtemi.';inin in A. ClnlZua

T he leaves were pulve rized . Hexane extracts were prepared fro m O. I g leaf material and/or leaf + inflo rescence material ( 1999, last ha rvest) . Concentration of a rtemi s in in was determined by HPTLC6

. T he y ie ld and qua lity of A rtemisia annua cv. Ieevanraksha leaves obtained duri ng the year 1999 and 2000 have bee n shown in Tab le I .

Bench Sca le Studies

Benc h scale experi men ts were first carried out fo r the deve lop men t of an improved process fo r the ~; e l ec tive isolation of a rtemis inin . Optim izati on of the extrac ti o n paramete rs like contact t imes of different so lvents with the raw materi a l, extract ion temperature and mini mum number o f so lven t was hes required to ex trac t the max imum quanti ty of artemisi nin was carri ed out. T he process of separat ion of the fat and other undes ired materia l and the se lec ti ve isolation of a rtemi s inin from the c rude ex tract by the process of iiqui d-li qu id ex tract ion using so lve nts was al w stud ied . T he purif ied extract was chromatographed

Table I- Yield and qua lity of A r1 e llliol ia C/II I/UC/ cv. l eevanra ksha leaves obta ined from ratooncd crops in Indo­

Gangetic agro-cl i mate in the year 1999 and 2000

Cropping Harvest Crop age at Dry leaf Artemisinin in

year nu mber the lime of yield, harvested

harvest, q/ha leaves,

d per cent

1999-99 84 IS.5 0.64

2 155 13. 1 0.99

3 7..27 33.9 0.53

1999-2000 97 23.8 0. 67

2 145 12. 1 0.g9

IgO 15.7 1.06

4 205 18.6 1.09

over si lica ge l to get pure artemi sinin . The results and data obtained fro m the bench scale studies were evaluated and scaled up to the pilot scale leve l.

Determination of So lubility of A rtemisinill

For the des ign of ex trac tion equipment involving artemis inin , accurate data on its sol ubility in vari ous solvents was required . As th e da ta was no t avai lable in the literature it was determined experime nta ll y us ing the two solvents empl oyed in the improved patented technoiogy . As so lubility varies with temperature, data was co llec ted at 20-

SO°C at SoC interva ls. T he data is presented in Tab le 2 . It can be noti ced from the data that the so lub il ity of artemi s inin is much hi gher in the po lar so lvent, whi ch great ly favo urs the mass transfer of a rtemi s ini n during the liquid- liquid part ioning of the ex trac t f rol11 non-polar to po lar phase.

Pilot Scale Studies

O ptimi zati on stud ies we re carri ed out to improve the se lec tive extracti on of a rte mis inin from Artemisia annua herb on pilot p lant scale. T he extraction of the dried herb was carri ed out in a 40 ka

sta inless stee l so lvent ex trac ti on pl ant. T he se l ec t iv~ parti oning of artemis inin from the c rude ext ract was carri ed out in a countercurrent liquid-liqu id ex traction column. L iquid-liquid ex trac ti on parameters li ke feed flo w rates , agitator frequency and a mplitude were optimi sed to ac hieve complete mass transfer of the artemi s in in in a s ing le pass . Solvents were recovered

under vacuum at less than SO°c. For the fin a l step of puri ficati on of the extrac t by s ilica ge l co lumn chromatography the rati o of s ilica ge l to extrac t weigh t was standardi sed .

Table 2 - Solu bi lily of artcmisinin

S No Temperature, Solubili ty , gil 00 g DC

In non-polc,r In polar sol vent solvenl

20 O. i6 32.4 1

2 25 0.3 1 36.93

3 30 0.45 38.59

4 35 0.57 5lJ.65

5 40 0.60 85.93

6 45 0.83 92.4 :

7 50 0.9 1 98.55

TAN DON e / 01.: ISOLATION OF ARTEMISININ 459

Description of the Isolation Procedure and Processing Equipments

The extraction of artemisinin from Artemisia annua on pilot scale was carried out in three stages:

Stage 1- Solvent Extraction of Plant Material

After estimating the moisture content by drying a smail known quantity of the herb in a hot air oven at 60°C for 6 h, the dried and powdered Artemisia annua herb is filled in the drug holder of the solid­liquid extraction plant. The main equipments of this pilot plant consist of a 40 kg capacity drug holder and a solvent reboiler jacketed kettle by which the solvent is distilled. The solvent losses are minimized in the process by providing two shell and tube condensers for recovery of sol vents. The extracting non-polar solvent designated as Solvent' A' is transferred from solvent storage tank to drug holder so as to completely immerse the herb. Required contact time is given for first percolation. The extract is then drained into the reboiler kettle where the solvent is distilled and recycled back to process the herb. Similarly, five percolations are given for complete ex traction of the herb. The solvent is then recovered back from the reboiler kettle and also from the marc into the storage tank.

Stage 2-Liquid-liquid Extraction

The crude extract obtained from the solvent extraction plant is further subjected to liquid-liquid extract ion for removal of undesired constituents. A polar solvent designated as Solvent 'B', selected after bench-scale studies is taken for the liquid-liquid extraction . Both the sol vent phases are transferred to feed vessel s of the reciprocating plate type countercurrent liquid-liquid ex traction column. The glass column of he ight 2700 mm and two separating compartments of 4 L capacity each and consisting of perforated disks was used for the selective isolation of the artemisi nin from the non-polar to the polar solvent. The flow rate of the extract and the column agitator frequency are maintained so as to achieve the complete mass transfe r of the artemisinin and to avoid f looding of the column. The liquid-liquid extraction is carried out for 3 h with continuous collection of the ex tract and the raffinate. Moisture is removed from the polar solvent by addi tion of a desiccant. Both the phases are then concentrated and solvents are recovered ill a vacuum concentralor to prevent any degradation of artemisinin .

Stage 3-Column Chromatography

The extract obtained after concentrating the polar solvent is column chromatographed for the purification of artemisinin. The extract is purified in a stainless steel preparative type column with capacity of holding 100 kg silica gel. The slurry of the extract is prepared using column s ilica gel and dried under vacuum. Fresh silica gel is loaded into the chromatography column with slurry and is eluted with solvents. The polarity of the eluent is gradually increased tiil fractions substantially rich in pure artemisinin is eluted from the siiica gel. Several fractions of 40 L are collected with simultaneous monitoring by TLC. The solvents are simultaneously recovered by distillation. The concentrated fractions are allowed to cool down to crystallise and are then filtered to obtain the artemisinin. The purity of the artemisinin and aiso the artemisinin content of the raw materials was estimated by the HPTLC method5

.

Results and Discussions

In 1999-2000, 25 batches (E I-E25) were extracted, while in the year 2000-200 I the herb was extracted in 26 batches (A I-A26) . The tota l artemisinin content was evaluated for each ex traction batch separately. The dry weight of plant material being loaded in the solvent extraction plant was calculated from the moisture content data . The average moisture in dry leaves was 5.5-6.5 per cent. The average artemisinin content in the first year crop was around 0.64 per cent while in the second year it increased to 0.86 per cent. The data on moisture and artemisinin contents and recovery are given in Table 3 and 4. Further, liquid partitioning and isolation was carri ed out in three separate parts . The fi rst part consisted of extraction batches from E I- E9 (total dry pl ant material = 344.13 kg) and second part from batches EwE25 (total dry plant material = 6 10.50 kg) . The second year herb was collectively processed as third part for batch numbers . AI -A l () (total dry pl ant material = 846.56 kg) . The crude extract obtained from these batches cons:isting of (·'ats , waxes and artemisinin etc . was processed in liquid-li quid ex traction column for selective isolation of artemisinin. For the first part the liquid liqu id partitioning was carried out by combining :2 extract ion batches, the second part by combini ng 4 extraction batches while the third part was scaled up to handle 6 extraction batches simultaneousl y. The

460 J SCI IND RES VOL 62 MAY 2003

Table 3 - Arte l1li sinin recovery from A. ({ IlIl IlCl herb of 1999-2000

Batch Leaves loaded Moistu re in Artemisinin No . in extractor, leaves, by FWfLC,

klT <0 per cent per cent

EI 40.5 8.20 0.S2

E" 40.2 6.70 0.54

E\ 37.0 3.60 0.54

E, 37.0 5.07 0.61

E, 4 1.5 5.02 0.74

EI, 43 .2 7.00 0.6 1

E7 43.5 7. 3 1 0.70

E" 42. 1 7.83 0.6 1

E) 42.0 5.40 0.59

Total 367.0 56.1 3 5.76

Mean 6.23 0.64

Arte illi sin in recovered = I.S50 kg (0.54 per cent on dry weight basis) Efficiency of ex traction = 84.4 per ceni

Elil 40.{) 6.90 0.8 1

Ell 39.3 6.90 0.8 1

E, " 430 5.40 0.73

El.l 4 1.7 5.27 0.59

EI-l 40.0 :i25 0.63

E" 390 5.3 1 0.63

E'I> 43.0 6.08 0.5S

EI7 39.5 5.72 0.49

E lx 40.6 5. 54 0.72

E,,) 41.4 5.8 1 0.67

E,II 40.4 5.67 0.60

E" 40.0 5.50 0.64

E"" 40.8 6.54 0.67

E".1 4 1.0 5.8 1 0.72

E2-l 42.0 5.90 0.60

E" 37.0 6.52 0.54

Tota l 648.7 94. 12 10.43

Mean 5. 8') 0.65

Arlemisin in Recove l'ed = 3.3')0 kg (0.56 per cent a ll dry weight basis) Effi ciency 01' extrac ti on = 85.9 per cent

tota l extract obtai ned afte r the liqu id part ioning from the po lar ph ase was 8.7 kg for the fi rst part, 13 .5 kg for the second and 25 .0 kg for the third part, respect ive ly. The production data fo r iso lation of artemis inin f rom A. (f/lIUW at pi lot plant is given in Tab le 5. By compar ing the processing data of the three separate parts it can be observed that the effi c iency of ex trac tion process improved with the increase in the herb being processed from 84.3 to 86.3 per cent. From the economi c angl ~ the weight of silica gel consumed/kg of artemi sin in produced and

Table 4 - Artemisi nin recovery frolll A. ({1l1l 1/0 herb of 2000-2001

Batch Leaves loaded Moisture in Arlemisin in by No . in Extracto r, leaves, HPTLC,

k(T "

per eent per cent

AI 32.5 8.48 0.70

A, 30.0 8. 14 0.95

A, 35.0 5.78 1.00

A-l 32.5 6 .1 0 0.63

As 3 1.4 6.3 1 1.00

AI, 30.0 6.2 1 093

A7 30.0 6.30 1.00

Ax 30.5 5.62 0. R5

A ) 38 .S 6. 28 0.')0

AlII 38.0 6.40 1. 00

All 36.0 5.92 0.90

AI" 39.0 6.30 0.63

Au 37.6 6.54 0.98

AI -l 39.0 5.4') 0')0

A I.l 36.0 5.92 088

A'I, 38.0 5.83 0.90

AI7 37 .0 7. 16 003

A,x 35 .6 6 . ~9 0.90

A , ~ 37.0 6.70 085

A211 34.0 5.90 0.78

A2I 40.0 5.S0 1.00

A22 38 .0 5.80 1.00

A2.1 320 5.48 0.63

A2., 3 1. 6 5.86 0.95

A" 3 1.6 5.84 0.80

A2(l 32.4 6.65 0.63 Total 903.2 163.2 22.32

Mean 6.27 0.85

Artemi sinin recovered = 6.280 kg (0.74 pe r ce nt 011 dl'Y weight basis) EtTiciency of extraction = 86.3 per cent

the ratio of weight of s ili ca ge l to extrac t a lso decreased fro m 25.40 to I 1. 82 kg, thereby reduc ing the Gvera ll work ing cost. By increasin g the batch s ize , the solven t losses a lso reduced s ignificantl y. T he puri ty of the artem is in in reco vered was 97-98 percent.

Conclusion

CIMAP has successfull y developed a hi gh yie ldin g va ri e ty of Artemisia {l/lI71W wh ic h is sui table for culti vation in north In di an p la in s. In view or the

large potenti a l requi rement for the ant i-ma lar ia l a rtemis inin deri vati ves in Ind ia , hav ing Artel1lisi({ (/JU/ua variety suitab le for cu lt ivation in the sub

TANDON el at.: ISOLATION OF ARTEMISININ 46 1

Table 5 - Overall prod uction data for arlcmi si nin rrom A. aannlla in the pilot plant

Parameter Part I Part II Part II I (E I- E~) (E IO-E2,) (A I-A2(,)

Plant Material , kg 343.53 6 12.78 850.60

Crude Extract, kg 20.6 1 30.63 58.40

No n-polar solvent losses 35 48 79 during extraction , L

No n-polar so lvent ra ffin ate 11 .6 16.86 33. 1 (after I iq- I iq ) , kg

Po lar so l vent ex tract 8.7 13.5 25.0 (a rter liq-liq ), kg

Po lar so lvent losses during 20 32 42 liquid liqu id ex tractio n, L

So lvent losses during 80 11 0 135 column chromatography, L

Vo lume of each fract ion 40 40 40 co ll ected, L

To tal no. of rract ions 80 90 11 0

Artemi sin in recovered . kn ~

1. 850 3.390 6.280

Efficiency of extrac ti on, 83.9 85.4 86. 3 per cent

Silica gel consumed , kg/kg 25.40 22.12 11.94

No n-polar so lvent 62. 16 46.60 34.07 consumcd, U kg

Po lar so lvent co nsumed, 10.8 1 9.43 6.68 Llkg

tropical c limate of north India is very s ignificant. The process ing of the new vari ety of A.ollll lla has been standardised on p ilot plant scale , which has generated suffic ient data required for the des ign of commercial sca le plants. The improved process is hi ghly effici ent and economica l as mo ~,t of the solvents and adsorbents being used in the process are being recovered and reused. All these advan tage are of

significant economic value for large-scale production of antimalarial drug artemi sinin .

Acknowledgments

The authors are grateful to the Department of Biotechnology for providing financ ial assistance. The authors are thankful to M S Siddique, lamil Ahmad , A R K idwai , Ram Lakhan and S C houdhary for providing technical ass istance during the pilot plan t operations. The autho rs are al so thankful to Dr M M Gupta for carrying out the anal ys is of the plant samples and D r S K Gupta fo r prov iding the plan t material fo r the pilot scale studies.

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